key: cord-0004837-02wem2u3
authors: Wada, R.; Fukunaga, Y.; Kondo, T.; Kanemaru, T.
title: Ultrastructure and immuno-cytochemistry of BHK-21 cells infected with a modified Bucyrus strain of equine arteritis virus
date: 1995
journal: Arch Virol
DOI: 10.1007/bf01322744
sha: 5d39c14adea5f79fa8fc27eedb111211146d660a
doc_id: 4837
cord_uid: 02wem2u3

Morphogenesis of a modified Bucyrus strain of equine arteritis virus (EAV) in BHK-21 cells was studied. Bacillary tubules were first detected in the cytoplasm 8 h after infection, and mature virions 79 to 122 nm in diameter, 101 nm on average, were mostly observed in the cisternae of the rough endoplasmic reticulum (RER) at 12 h or later. They had isometrical cores and morphological subunits in the outer layer. Budding occurred from the RER and the outer nuclear membrane, but not from the cell surface. Structural linkage was detected between the tubule and the virus core. Aberrant strands were occasionally demonstrated within the nucleus 12 h after infection, and immunofluorescence and immunogold labeling revealed viral antigen also in the nucleus.

Equine arteritis virus (EAV) has been classified into the Togaviridae family [22] on the basis of the virion size and morphology [23] . Recently, however, the completely sequenced EAV genome revealed that the virus is of the coronavirus superfamily [7] .

The EAV virion was shown to be a spherical particle approximately 60 nm in diameter, consisting of an icosahedral core of 35 nm in diameter surruonded by an envelope [t, 9,14, 18] . Because of difficulty of treating virions not only in vivo [3, 5, 9] but also in vitro [1] , few works have been published on the morphologic characterization of EAV.

In the present study a mutant virus showing higher growth on BHK-21 cells than the parental Bucyrus strain of EAV [11] was examined for the fine structure, its morphogenesis and intracellular localization of viral antigen.

Confluent monolayers of BHK-21 cells grown in 25 cm 2 plastic dishes were rinsed with Eagle's minimum essential medium (Eagle's MEM) and they were infected with a mutant virus, the 1174 R. Wada et al.

modified Bucyrus (mB) strain of EAV [19] , at a multiplicity of infection of 10. After incubation at 37 °C for 1 h, the infected cultures were rinsed with Eagle's MEM and then received 5 ml of Eagle's MEM with 10% fetal calf serum (maintenance medium). At 4 to 48 h of incubation at 37 °C, samples were taken, and stored at-70 °C for the determination of virus yield and fixed for transmission electron microscopy (TEM). The virus titer was determind by plaque assay, as described by Fukunaga et al. [12] .

Monolayer cultures of BHK-21 cells grown on coverslips in perti dishes were washed twice with 0.01M phosphate-buffered saline (PBS), dried and fixed with aceton at-20 °C for 10 rain. They were treated with mouse monoclonal antibody mB 1A3 specific for structural protein of EAV [17] , which was used as undiluted hybridoma supernatant. After washing 5 times with PBS, the cultures were stained with fluorescent isothiocyanate conjugated goat ,/-globulin to mouse ~-globutin at 37 °C for 60 min. After washing with PBS and mouting in SlowFade (Molecular Probes, Inc., OR, USA), the stained samples were examined under the fluorescent microscope.

Infected cells were washed twice with 0.05M sodium cacodylate buffer (pH 7.4), and fixed at 4 °C for 1 h in 2% glutaraldehyde and 0.05M CaC12 in the same buffer. Samples were then washed twice in the buffer solution, and postfixed at 4 °C for 30 min in the buffer with 1% osmium tetroxide. Cells were then removed from the plastics using a rubber policeman, pelleted by centrifugation at 12 000 rpm for 5 min, embedded in 1% noble agar, dehydrated in an ascending ethanol series, and embedded in Poly/Bed 812 (Polyscience Inc., PA, USA). Ultrathin sections were made and stained with uranyl acetate (2%, w/v) and Reynold's lead citrate. They were examined using a Hitachi H-7100 electron microscope at 75 kV.

For immunogold labeling sections were mounted on a 200-mesh nickel grid, which were dipped in PBS containing 0.5% bovine serum albumin for 5 min, and then incubated with monoclonal antibody mB 1 A3 at room temperature for 60 min. After washing in PBS, the grid was incubated with goat antibody to mouse IgG, which was labeled with 5 nm gold particles (Biocell Co., U.K.) at room temperature tbr 60 min. After washing in PBS and rinsing in distilled water, the grid was counterstained with uranyl acetate and Reynold's lead citrate.

Viral antigen was first detectable at 8 h after infection and the growth reached a peak at 14 h (Fig. 1) . Virus antigen first appeared as fine granules in the cytoplasm of a few cells. Specific fluorescence significantly increased 10 to 12 h after infection in the perinuclear region of the infected cells (Fig. 2a) as well as within the nuclei at 12 h or later (Fig. 2b) . Later, the distribution of viral antigen became more generalized within the cytoplasm, although there was some aggregations within the nuclei. No virus-specific fluorescence was seen in uninfected cells and those treated with normal mouse serum and without monoclonal antibody mB 1A3. 

At 4 to 6 h after virus infection, the cells did not show any morphological changes. At 8 to 10 h an accumulation of ribosomes was shown and a few aberrant strands and short bacillary or tubular branching structures appeared in the cytoplasm. The tubules increased in number thereafter, especially in the vicinity of the nucleus (Fig.  3a) . They were more than 200 nm long and on average 42 nm in width, ranging 32 to 52 rim. Aberrant strands were observed frequently in the nucleus at t2 h or later after infection (Fig. 4c) . Virus particles first appeared at 12 h after infection in the cisternae of the rough endoplasmic reticulum (RER) as well as in the perinuclear space. At 14 h virions increased in number in a larger number of cells (Fig. 3 a) . They were usually spherical or oval 79 to 122 nm, 101 nm average (n=48), in diameter, with isometrical cores 37.8 nm in diameter(n=35). The core had electron-lucent outer layer and dense center with uniformly high opacity (Fig. 3b ) or electron lucency (Figs. 3c, e). Through cross-sections of the outer layer of the virion, a grape-like structure seemed to be subunits 13 to 20 nm in diameter around the core (Fig. 3c) . Some virions had an envelope with tiny surface projections (Fig. 3b) . Budding of the virus was occasionally observed at the cytoplasmic membrane (Figs. 3d-f ). In some budding particles a structural linkage between the tubules and cores of the maturing virions was demonstrated (Fig. 3g, h) . On the other hand, other small sized particles about 40 nm in diameter, seemingly in a process maturation, were sometimes observed in the cytoplasmic vesicles (Fig. 3i ). Some of them had a very electron-lucent outer layer. Maturation by budding across the cellular membrane could not be found.

Tubules (Fig. 4a) and virus core (Fig. 4b) in the cytoplasm as well as strands (Fig. 4c) in the nucleus were labeled with immunogold staining.

EAV has been reported to be morphologically similar to arboviruses [1, 2, 14, 15] and classified into the Togaviridae family [10, 13, 22] while morphogenesis of EAV remains unclear. Recently, however, den Boon et al. [7] reported that EAV is evolutionally related to the coronavirus superfamily. A new virus family, the Arteriviridae, which includes the lactate dehydrogenase-elevating virus, lelystad virus and simian hemorrhagic fever virus, has been proposed by Plagemann et al. [20] .

EAV are 60 nm in diameter with 12-15 nm ring-like subunits on the surface, having a double membrane structure in an electron-dense core 25 to 35 nm in diameter and possessing an electron-lucent outer envelope [9, 10, [13] [14] [15] 18] . Ringlike subunits may exist in the envelope [13, 15] . They also have tiny spikes on their surface [14, 15, 18] . Those structural characteristics were commonly revealed in both purified virus samples [13, 14, 18] and sections [1, 9, 14, 18] .

In the present study the mature virions of the mB strain of EAV were 79 to 122 nm in diameter, 101 nm on average, being significantly larger than those reported before. The outline of the mature virions in the RER cisternae was always indefinite, and some of them had distinct surface projections like coronavirus. The mature virions appeared to have subunits in the electron-lucent outer layer. Each subunit was spherical 13 to 20 nm in diameter. These findings suggested that the ring-like subunits were viral capsid components produced inside of the envelope at the budding sites.

Other small sized particles with an approximate diameter of 45 nm were sometimes observed in the cytoplasmic vesicles. They were similar to the typical EAV particles with a thin outer layer around the core [1, 9] . There might be possibilities that: (1) the virus cores broke through a damaged endoplasmic membrane into the vesicles; (2) the outer layer of the small particles was not properly stained; (3) the small particles were incomplete virions without outer subunits. Maturation and budding of the virus appears to occur at RER and perinuclear membrane, but not at the cell membrane. A large number of tubules, assumed to be specific structures related with the replication of EAV in vivo [9] and in vitro [2] , were observed in BHK-21 cells infected with the mB strain of EAV. In this study, a structural linkage was detected between the tubules and the virus core. These tubules reacted with immunogoldlabeled antibody to EAV. Similar tubules have been described also in infections with lactate dehydrogenase-elevating virus [21 ] , mouse hepatitis virus [8] and bluetongue virus [4] , while larger number of tubules appeared in infection with the mB strain of EAV.

No ultrastructural or immuno-cytochemical change in the nucleus has been observed previously [2, 6, 16, 18] . In this study, however, EAV-specific ultrastructural and immunofluorescent findings were shown in the nucleus. By immunogold labeling of thin sections, antigen was detectable not only on the cytoplasmic tubules but also on strands in the nucleus. These findings suggested the same viral components were present in different forms in both sites. It could not be determined whether the strands in the nucleus were precursors of tubules or they represented in dead-end stage of the virus wandering in the nucleus, since no morphological transition was observed.

Electron microscopic characterization of equine arteritis virus

Equine arteritis virus: ferritin-tagging and determination of ribonucleic acid core

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The pathogenesis ofbluetongue virus infection ofbovine blood cells in vitro: ultrastructural characterization

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Fluorescent and electron microscope studies of equine arteritis virus (Abstr.)

Equine arteritis virus is not a togavirus but belongs to the coronavirus-like superfamily

Cell tropism and expression of mouse hepatitis viruses (MHV) in mouse spinal cord cultures

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The authors gratefully acknowledge T. Yamakawa and S. Shibata for their excellent technical assistance.

Authors' address: Dr. R. Wada, Epizootic Research Station, Equine Research Institute, The Japan Racing Association, 1400-4, Shiba, Kokubunji-machi, Shimotsuga-gun, Tochigi 329-04, Japan.Received December 28, 1994