J. gen. Virol. (1988), 69, 805-815. Printedin Great Britain
805
Key words: BTV/capsid proteins/immunogold electron microscopy
Ultrastructural Distribution of the Major Capsid Proteins within
Bluetongue Virus and Infected Cells
By A L E X D. H Y A T T * AND B R Y A N T. E A T O N
Commonwealth Scientific and Industrial Research Organization, Australian Animal Health
Laboratory, P.O. Bag 24, Geelong, Victoria 3220, Australia
(Accepted 7 January 1988)
SUMMARY
Core proteins VP7 and VP3 have been localized in bluetongue virus (BTV) and
BTV-infected cells by immunoelectron microscopy. Gold-labelled monoclonal antibodies to VP7 gave intense labelling with purified BTV core particles and weaker
labelling with both directly visualized viral particles, which require no purification,
and purified virus particles. It is believed that VP7 is, in a small number of viruses,
accessible from the outer surface. The intensity of labelling by anti-VP7 antibodies was
markedly increased by treatment of the virus with methanol. Intracellularly, VP7
antibodies also reacted with virus-like particles which appeared to be leaving virus
inclusion bodies (VIB), the presumed site of virus synthesis and assembly. These
antibodies also reacted with virus-like particles which were bound to the cytoskeleton
and did not appear to be virus cores because they also reacted with gold-labelled
antibody to the outer coat protein VP2. VP3 was not detected immunologically in either
virus or core particles nor in cytoskeleton-associated virus-like particles suggesting an
inner core location. VP7 and to a lesser extent VP3 were localized within the matrix of
VIB. Virus tubules, a major structure found in infected cells and known to contain the
non-structural protein NS1, were found to react with antibodies to both VP3 and VP7.
INTRODUCTION
The genus orbivirus is one of six in the family Reoviridae and is divided into 13 serogroups on
the basis of group-reactive tests. The bluetongue serogroup comprises 24 virus serotypes which
have been identified using virus neutralization tests. Bluetongue virus (BTV) contains 10
dsRNA segments (Verwoerd et al., 1979; Gorman et aL, 1983) each of which codes for a single
protein (Grubman et al., 1983; Mertens et al., 1984). Morphologically, the virus consists of a
core, composed of 32 capsomeres arranged in icosahedral symmetry, surrounded by an outer
fibrillar coat (Bowne & Richie, 1970; Verwoerd et al., 1972). The outer coat layer is composed of
two major structural polypeptides (VP2 and VP5) of which VP2 is the dominant BTV serotypespecific antigen (Huismans & Erasmus, 1981 ; Appleton & Letchworth, 1983). The core contains
two major (VP7 and VP3) and three minor (VP 1, VP4 and VP6) structural polypeptides. Recent
evidence indicates that VP7, the major bluetongue serogroup-reactive antigen (Huismans &
Erasmus, 1981 ; G u m m & Newman, 1982), is the major constituent of the capsomeres on the
surface of BTV core particles (Huismans et al., 1987). VP3 has aso been shown to contain groupreactive sites (Huismans & Erasmus, 1981).
Cells infected with BTV contain two major non-structural proteins (NS1 and NS2) in addition
to the seven structural proteins outlined above (Huismans & Els, 1979; Huismans & Basson,
1983). The cytoplasm of BTV-infected cells is characterized by the presence of fibrillar virus
inclusion bodies (VIB) and virus-specified tubules (VT) (Lecatsas, 1968). VIB are believed to be
the site of virus morphogenesis. Tubules contain predominantly NS1 (Huismans & Els, 1979)
and their function in replication is obscure. We have recently used monoclonal antibodies to
NS2, VP2 and NS1 in immunofluorescence and immunogold procedures to localize virus
0000-8108 O 1988 SGM
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A. D. HYATT AND B. T. EATON
proteins in BTV-infected cells and cytoskeletons (Eaton et al., 1987, 1988). In the experiments
reported here we have used monoclonal antibodies to locate the major core proteins VP7 and
VP3 in infected cells and cytoskeletons and in virus and core particles of BTV. The results
confirm the recent finding by Huismans et al. (1987) that VP7 is the major surface component
of core particles. In addition, the data indicate that VP7 is present within VIB, is found in
association with VTs and is weakly expressed at the surface of some virus particles.
METHODS
Virus, cells and monoclonal antibodies. An Australian isolate of BTV-1 (CSIRO 156) (St. George et al., 1980) was
plaque-purified and used to infect SVP cell monolayers at a multiplicity of approximately 5 p.f.u, per cell as
previously described (Eaton et aL, 1987). Virus and core particles were purified as described by Mertens et al.
(1987). Virus in the cytoplasm of infected cells was harvested at approximately 36 h post-infection (p.i.) by treating
infected cells with 1 ~ NP40 in STM (10 ml~l-Tris-HC1, 0-15 m-NaC1, 5 mM-MgCI~, pH 7.4). Nuclei were removed
by centrifugation and the cytoplasmic supernatant was pelleted through 4 0 ~ sucrose at 39000 r.p.m, in a
Beckman SW41 rotor, for 90 min at 4 °C. Virus was resuspended in STM and the preparation referred to as
cytoplasmic (crude) virus.
All of the monoclonal antibodies except 20H(2) were derived from subcloned hybridoma cell lines and obtained
from J. R. White (unpublished data) as Protein A-purified mouse ascitic fluid. 20H(2) was a hybridoma cell
supernatant. Monoclonal antibodies (MAb) used in this study reacted with VP7 (20E9/B7/G2) and (20A11/10),
VP3 (7A4/A11), NS2 [20H(2)], NS1 (20E6/A4) and VP2 (9J3/G3). Monoclonal antibody (20E9/BT/G2) and MAb
(7A4/A11) precipitated VP7 and VP3 respectively. MAb (20A11/10) reacted with VP7 in Western blots and MAb
(9J3/G3) precipitated VP2 and neutralized the virus. The antibodies were conjugated directly to either 6 nm or
14 nm gold particles as described by Slot & Geuze (1985). Monoclonal antibodies are referred to hereafter as MAb
ctVP7, c~VP3,~NS2, ~VP2 and ctNS1. Except where otherwise stated the ~VP7 antibody utilized was 20E9/B7/G2.
Antibodies conjugated to gold are designated as follows, Au MAb c~VP7 and Au-MAb ~VP2.
Immunofluorescence. Cells for immunofluorescence studies were grown on glass coverslips and infected with
BTV at an m.o.i, of 5 p.f.u, per cell. Mock-infected and BTV-infected cells (18 to 20 h p.i.) were washed in
phosphate-buffered saline (PBS) and treated sequentially with methanol and acetone at - 20 °C and then airdried. Viral antigen distribution was determined using appropriate antibodies which were localized with
biotinylated anti-mouse antiserum and fluorescein-streptavidin (Amersham). Primary incubations in double
labelling experiments were carried out with fixed cells using MAb ~VP7 and fluorescein-conjugated anti-mouse
IgG serum. Secondary incubations utilized biotinylated MAb ~NS2 followed by Texas red-streptavidin
(Amersham). Cytoskeletons were prepared by treatment of cells with PBS containing 1 ~ NP40 and 0-1~
glutaraldehyde. Following two washes with PBS and PBS containing 1 ~ bovine serum albumin (BSA), (PBSBSA), and without drying, cytoskeletons were probed with MAb c~VP2 or MAb ~VP7 as described above. To
enhance the fluorescence achieved with MAb ~VP7 and to demonstrate activity with MAb ~VP3 and MAb ~NS1,
cytoskeletons were treated with methanol at - 2 0 °C prior to washing with PBS-BSA.
Negative contrast immune electron microscopy (NCIEM). Cells grown and infected on electron microscope grids
(grid cell cultures, GCC) as described by Hyatt et al. (1987) were rinsed three times in PBS. Cells were either fixed
for 2 min in PBS containing 0.1 ~ glutaraldehyde or cytoskeletons were generated from them and fixed in PBS
containing 0.1 ~ glutaraldehyde and 1 ~ NP40. Cells and cytoskeletons were washed three times (5 min per wash)
with PBS. In experiments involving the use of MAb aVP7 and MAb ~NS1, cytoskeletons and cells were treated
with methanol at - 2 0 °C for 2 min. Cells and cytoskeletons were washed six times with PBS BSA prior to
incubation with the appropriate antibody for 1 h at 37 °C. Incubation with MAb which was not conjugated
directly to gold was followed by six washes with PBS BSA and a further incubation with Protein A-gold (prepared
as described by Slot & Geuze, 1985) at 37 °C for 1 h. Gold-labelled viruses and cytoskeletons were rinsed six times
in PBS-BSA, post-fixed and stained or critical point-dried as described by Hyatt et al. (1987). Double labelling
experiments involved primary incubations with Au-MAb ctVP2 (14 rim) as described above followed by a further
incubation with Au MAb ~VP7 (6 nm) at 37 °C for 1 h prior to washing and fixation.
NCIEM was also performed on purified virus particles and cores adsorbed onto parlodion-filmed copper grids
for 2 min. The procedure used for labelling and staining was identical to that outlined above. Preparations were
stained with 2 ~ phosphotungstic acid and buffered to pH 6.5 with KOH (KPT).
Thin section, post- andpre-embedding immunogoM labelling. To probe post-embedded cells, monolayers in plastic
tissue culture flasks were infected with BTV at 5 p.f.u, per cell and at 20 h p.i. scraped from the flasks. Infected
cells were fixed with 0.25 ~ glutaraldehyde in 0.1 ~ cacodylate buffer (300 mmol/kg, pH 7.2), rinsed in cacodylate
buffer, dehydrated in ethanol and infiltrated and embedded in Lowicryl K4M (4 °C to - 25 °C).
Polymerization occurred at - 2 5 °C overnight under u.v, irradiation. Sections were cut onto nickel grids,
washed with PBS-BSA (six times, 3 min each), incubated with normal rabbit serum (1 h, 37 °C) followed by MAb
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Distribution of bluetongue virus proteins
807
~VP7, MAb ~NS2, MAb ~VP3 or MAb c~VP2(1 h, 37 °C). After six 3 rain rinses in PBS-BSA sections were
incubated with biotinylated anti-mouseantiserum (1 h, 37 °C) followedby six further 3 min PBS-BSA rinses and
streptavidin-gold (1 h, 37 °C) (Amersham). Sections were stained as described by Eaton et al. (1987).
Specimens to be labelled before embedding were extracted with 0-1~ glutaraldehyde and 0.1 ~ NP40 and
labelled as described above for NCIEM. After final incubations and washes, the virus-infected cells were fixed
and processed as for conventional samples (Eaton et al., 1987).
Preparations were viewed in a Hitachi H600 scanning transmission electron microscope at 50 and 75 kV.
RESULTS
Analysis of B T V particles by N C I E M
Cells grown on electron microscope grids were infected with BTV and 24 h later viruses
released from the cells and adsorbed to the grid were probed with A u - M A b ~VP7. These viruses
(termed native viruses) reacted weakly but specifically with the antibody (Fig. 1a). Not all
viruses were labelled and those which were possessed only one or two gold probes. Viruses
isolated from the crude cytoplasmic fraction of infected cells and partially purified by
sedimentation through a sucrose column gave more intense labelling when probed with
Au-MAb ~VP7 but differed morphologically from native viruses in that capsomeres could be
observed underlying the outer fibrillar coat (Fig. 1 d).
A significant proportion of VP7 in infected cells exists in a soluble form (Eaton et al., 1987).
To eliminate the possibility that viruses may be contaminated with adventitiously adsorbed
VP7, BTV was purified as described by Mertens et al. (1987) and probed with Au-MAb ~VP7.
Morphologically, these viruses possessed the outer fibrillar coat and reacted weakly with
antibody. The results (not shown) were similar to those shown in Fig. 1 (a). The intensity of
labelling by Au-MAb ~VP7 was increased by treatment of native, cytoplasmic or purified virus
particles with methanol (Fig. 1 b). In order to confirm that Au-MAb ~VP7 was binding to
complete viruses, G C C were incubated with Au-MAb ccVP2 and both Au-MAb ~VP2 (14 nm)
and Au-MAb ~VP7 (6 nm). The results (Fig. 2) show that such extracellular particles are
labelled not only with Au-MAb ~VP2 but with Au-MAb ccVP7. It should be noted that although
methanol treatment of the virus increases c~VP7 activity we have found it to decrease c~VP2
activity; hence the decreased number of Au-MAb ~VP2 probes depicted in Fig. 2 (inset).
Unlike complete viruses, purified BTV core particles labelled intensely with Au-MAb ~VP7
in the absence of any unmasking agent (Fig. 3). The identity of the particles as cores was
confirmed by the presence of conspicuous capsomeres. When purified virus and core particles
were incubated with MAb c~VP3 and Protein A-gold, no labelling occurred suggesting that
either the VP3 epitope underlies VP7 in BTV cores or the VP3 epitope is not presented to the
antibody in an appropriate conformation for reaction.
Fluorescence
Immunofluorescence studies using fixed BTV-infected cells showed VP7 to be distributed
throughout the cytoplasm. Both VP7 MAbs used in this study generated similar fluorescent
patterns in fixed cells. The pattern for MAb 20E9/B7/G2 is shown in Fig. 4 (a). Superimposed on
a faint fluorescence distributed throughout the cytoplasm are small discrete foci and large
circular bodies of fluorescence. Double labelling experiments (Fig. 4a, b) with MAb c~VP7
and MAb c~NS2 [NS2 is a component of virus inclusion bodies (Eaton et al., 1987)] show a
coincidence of labelling, indicating that the circular, fluorescent structures are VIB and that
they have a VP7 component. Immunofluorescent probing of detergent-generated cytoskeletons
with MAb ~VP7 (20E9/B7/G2) revealed an additional labelling pattern. As seen in Fig. 4 (c) the
cytoplasm contained many short fluorescent rods. MAb ~VP7 (20A10/A11) did not reveal these
structures. Immunogold procedures were utilized to determine the nature of the VP7-containing
structures associated with the cytoskeleton of BTV-infected cells.
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808
A. D. HYATT AND B. T. EATON
i(b)
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Fig. 1. Electron micrographs of BTV and core particles labelled with Au MAb c~VP7 (14 nm). (a)
Native viruses prepared from GCC. Preparation was negatively stained with KPT. (b) Viruses
prepared by GCC technique and treated, prior to labelling, with methanol. The preparation was critical
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, i •
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Distribution of bluetongue virus proteins
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Fig. 2. Electron micrograph of a negatively stained, VP2-1abelled GCC infected with BTV. Inset,
double-labelled (Au-MAb ~VP2, 14 nm; Au-MAb c~VP7,6 nm) virus. Cell (c) viruses (v). Bar markers
represent 100 nm.
Intracellular distribution of VP7 and VP3 by immunogold-labelled MAb
Due to the low antigenic mass of some viral proteins, particularly on the surface of embedded
sections, both pre- and post-embedding immunolabelling procedures were used in an attempt to
optimize the localization of intraceUular capsid proteins.
In preliminary experiments, cytoskeletons were prepared by NP40 treatment of BTVinfected cells on grids and were then probed with A u - M A b ~VP7. Labelling of virus-like
particles was intense after exposure of the cytoskeleton to methanol (Fig. 1 c) and weak without
methanol treatment (data not shown). Sections of A u - M A b ~VP7 pre-labelled cytoskeletons
revealed gold label, not only with virus particles throughout the cytoplasmic regions and at the
cell periphery but also with virus particles which were intimately associated with the VIBs
and/or fibrillar material morphologically similar to that constituting the VIB (Fig. 5 a). Lowicryl
sections labelled with M A b ~VP7 gave similar results but the degree of labelling was
substantially reduced (Fig. 5f). Sections of Au MAb ~VP2 pre-labelled cytoskeletons revealed
gold label associated with both cytoskeleton-associated particles (Fig. 5 b) and those apparently
leaving the VIB (Fig. 5 c). This suggests that these intracellular particles, although labelled with
A u - M A b ~VP7 are unlikely to be cores because they possess at least some of the outer coat
protein VP2.
point-dried and not stained, hence the variation in virus size between (a) and (b). (c) Cytoskeletal
filaments (0-associated viruses (v) directly labelled with gold probes. Preparation was critical pointdried and not stained. (d) Cytoplasmic viruses. Preparations were negatively stained with KPT.
Capsomere (arrow). Bar markers represent 100 nm (a, b and c) and 50 nm (d).
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Fig. 3. Purified BTV core particles labelled with
Au MAb ~VP7. The preparation was negatively
stained with KPT. Bar marker represents 50 nm.
Fig. 5. Electron micrographs of pre-embedded and post-embedded gold-labelled sections of BTVinfected cells. (a) Distribution of VP7 in an infected cytoskeleton. The cells were treated with methanol
prior to labelling, leading to a slight alteration in virus and tubule morphology. VIB (arrowhead),
viruses (v). (b) Association of VP2 with ¢ytoskeleton-associated viruses (v), virus-specified tubules (vt).
(c) Association of VP2 with viruses (v) emerging from a VIB (arrowhead). (d) Labelled Lowicryl section
showing the association of NS2 with VIB (arrowheads). (e, f ) Labelled Lowicryl section showing
distribution of VPT. Viruses (v), VIB (arrowhead). Bar markers represent 100 nm.
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(b)
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Fig. 6. Associationof VP3 and VP7 with virus tubules within cytoskeletons. (a) Cytoskeletonof GCC
infected with BTV and labelled with MAb c~VP3. The preparation was critical point-dried and not
stained. Virus tubules (vt), viruses (v). (b) Electron micrograph of pre-embedded, Au-MAb
20E9/B7/G2 (c~VP7)cytoskeleton. Virus-specified tubule (vt). Bar markers represent 100 nm.
Virus inclusion bodies within Lowicryl sections were identified by their ability to bind MAb
~NS2 (Fig, 5d). Labelling of the internal matrix of VIBs was also achieved with MAb c~VP7
(Fig. 5a) but not with MAb c~VP2 (data not shown). The gold probes associated with MAb ~VP7
did not appear to be associated with the dense, virus-like structures within VIBs. The data
suggest that VP7 is part of the VIB matrix.
Capsid proteins and virus tubules
Previous work has shown that antibodies to VP3 react with tubules in infected cells (Eaton et
al., 1987). Immunogold labelling of thin sections and cytoskeletons confirms the association of
VP3 with virus-specified tubules (Fig. 6a). No labelling with MAb c~VP3 was observed for virus
particles and only very weak labelling (one or two particles per VIB) was associated with the
VIBs. Fluorescence studies (Fig. 4e) suggested that VP7 may also be associated with tubules. To
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Distribution of bluetongue virus proteins
813
help clarify this relationship between VP7 and tubules, cytoskeletons were labelled with MAb
~VP7 (20E9/B7/G2) (Protein A-gold system). Preparations were subsequently processed for
thin sections and critical point-dried GCCs. Both procedures gave similar results in that VTs
were specifically labelled. Fig. 6 (b) unlike Fig. 6 (a) illustrates the results (MAb eVP7) obtained
by thin section electron microscopy.
DISCUSSION
Core proteins V P 7 and V P 3 have bcen localized in B T V and BTV-infected cells by
irnmunoclectron microscopy. The resultsindicatethat V P 7 isassociatedpredominantly with the
surface of the virus core or nuclcocapsid as reported by Huismans et aL (1987).The protein is
also found associated with VIBs, VTs and m a y bc acccssiblcat the surface of both nativc and
purified viruses from infected cells.
Viruses analysed by thc G C C technique as describcd by Hyatt et al. (1987), have not becn
subjectedto any forceswhich could damage the outcr coat.The abilityof some of these viruscsto
bind A u - M A b ctVP7 isthereforenot an artefactof viruspreparation but probably represcntsthe
in vivo state.The reaction of purified viruses with A u - M A b czVP7 strongly indicated that the
occasional expression of V P 7 on the surface of complete, native viruscs was not due to
adventitiouslyadsorbcd protcin.Thc possibilityexistshowever that some damage, albeitslight,
to the outer capsid layermay occur during sedimentation onto a sucrose cushion and so account
for thc low reactivityof A u M A b ctVP7. The number of labelledantibodies associatedwith thc
crude cytoplasmic preparation was grcater than that found with purificd virus particles.This
obscrvation may bc correlatedwith a more conspicious appearance of capsomcres which was
suggested to be mainly, ifnot entirely,composed of V P 7 (Huismans et al., 1987).Thc diffcrencc
in morphology and dcgree of labellingof cytoplasmic and purifiedvirusesmay well bc due to an
alterationin, or damage to, the structureof the outer coat of thc cytoplasmic virus induccd by
pelleting through sucrose and/or subsequent resuspension during partial purification. The low
labelling of native viruses and the progressive increase of labelling of virus particles as they gain
the morphological characteristics of cores, in the absence of methanol treatment, suggests that
VP7 may project beyond the core surface into the outer coat where in a small proportion of
viruses it may be able to interact with MAb aVP7. As stated above, it is unlikely that the outer
capsid layer observed in the GCC of viruses have been damaged, therefore the Au-MAb ~VP7labelled native viruses may indicate a heterogeneity in the virus population. Such heterogeneity
may be due to either the outer coat not covering the core in a consistent manner or the lability of
the outer coat proteins following release from infected cells.
The use of methanol significantly increased the intensity of VP7 labelling in native or
glutaraldehyde-treated viruses and virus-infected cells. Methanol, a known protein precipitant,
presumably causes a distortion of the normal protein conformation by sequestering water
molecules from the sphere of hydration of the virus proteins. Such distortion of the diffuse outer
coat of BTV (i.e. VP2 and VP5) would easily result in an increased exposure of the underlying
core protein VP7.
The absence of MAb c~VP3 labelling with virus and core particles was not unexpected.
Huismans et al. (1987) found that, following adsorption and penetration of infected cells, BTV
particles lose not only VP2 and VP5, but also VP7-containing capsomeres of the virus core. The
'sub-core' particles thus generated contained VP3. The immunoelectron microscopical studies
reported here support the conclusion that VP3 is present within core particles and underlies the
VP7-containing capsomeres. Specifically the results imply that the epitope involved in binding
to MAb ~VP3 is buried within the virus core although the possibility that the remainder of the
protein may be present on the core surface cannot be discounted. Recently, Huismans & Cloete
(1987) found that the gene coding for VP3 (RNA 3) was highly conserved among BTV serotypes.
In contrast, RNA 7, which codes for VP7, was not highly conserved. It is interesting to speculate
that the variability in RNA 7 may be due, in part, to the selective immunological pressure placed
on the potentially more exposed VP7 protein.
IntraceUularly, VP7 was localized to virus particles and VIBs. Collectively, the labelling of
infected cells with Au-MAb ~VP7 and Au-MAb ~VP2 shows that the core protein VP7 is
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A. D. HYATT AND B. T. EATON
incorporated into virus particles within VIBs. Furthermore the particles emerge from the VIB
with at least part of the outer coat VP2 component. A u - M A b ctVP2 fails to react with the
internal matrix of VIBs, thus the localized labelling of VP2 reported here may represent the sites
of VP2 synthesis. VP3 may also be present within VIB. Although the labelling of VP3 within
these structures is poor (approximately one or two gold particles per VIB) it should not be
dismissed as negative. Within viruses, VP3 is present in the core at about half the amount of the
other major core protein VP7 (Huismans et al., 1987). If this is representative of the VIB protein
content, and considering the level of VP7 labelling of VIB, the low but specific level of VP3
labelling is not unexpected. It is also possible that the optimum conditions for VP3 labelling
within VIBs have yet to be determined.
Virus-specified tubules, a structure common within BTV-infected cells, are known to contain
predominantly NS1 (Huismans & EIs, 1979). Results from this study show that VP3 and VP7 are
also associated with the tubules. The labelling is specific and of the cWP7 MAbs only
20E9/B7/G2 labelled the tubules, suggesting that the specificity is real and not a consequence of
protein contamination during cell extraction. The function of VTs still remains obscure but
perhaps the finding that they are composed of a mixed protein population may aid the eventual
elucidation of their role. The different labelling patterns observed with the two ~VP MAbs,
namely the ability of one to react with VTs suggests that they may react with different epitopes.
Furthermore as both MAbs react with native 'untreated' and 'treated' viruses it is possible that
at least two VP7 epitopes may on occasion be exposed on the surface of BTV particles.
Work on the distribution of other viral proteins such as NS2 and VP5 in viruses and cells is
continuing.
The provision of monoclonal antibodies by Mr J. White and technical assistance by Mr T. Wise, Mr G. Crameri
and Ms S. Brookes is gratefully acknowledged by the authors.
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