Antigenic variation in the bovine ephemeral fever virus glycoprotein was demonstrated using monoclonal antibodies and comparing a particular passage level of the BB7721 strain with (1) other Australian isolates of BEF virus, (2) the Beijing 1 strain of BEF virus, isolated in China and (3) batches of the BB7721 strain with different passage histories. Escape mutants of BEF virus were selected from cell culture and suckling mice, by growing virus in the presence of neutralising monoclonal antibodies. Escape frequencies were calculated for 14 monoclonal antibodies and an epitope map constructed of antigenic sites on the BEF viral glycoprotein which induce the production of neutralising antibodies

Cybinski, D. H.

St George, T. D.

University of Queensland eSpace


Paper 3.35
Antigenic Variation in the Bovine Ephemeral Fever Virus
Glycoprotein
D.H. Cybinski* and T .D. St George*
Abstract
Antigenic variation in the bovine ephemeral fever virus glycoprotein was demonstrated using
monoclonal antibodies and comparing a particular passage level of the BB7721 strain with (1)
other Australian isolates of BEF virus, (2) the Beijing 1 strain of BEF virus, isolated in China
and (3) batches of the BB7721 strain with different passage histories. Escape mutants of BEF
virus were selected from cell culture and suckling mice, by growing virus in the presence of
neutralising monoclonal antibodies. Escape frequencies were calculated for 14 monoclonal anti-
bodies and an epitope map constructed of antigenic sites on the BEF viral glycoprotein which
induce the production of neutralising antibodies.
RABIES viruses isolated from different animal
species in various parts of the world were formerly
considered to be closely related (Wiktor and Clarke
1973). However, more recent studies using mono-
clonal antibodies against the nucleocapsid protein
and the glycoprotein of the virus have provided
evidence of antigenic differences among several
strains of rabies virus (Wiktor and Koprowski 1980).
It has been suggested that these differences might
have been responsible for occasional failures in
postexposure vaccination of rabies patients. Further-
more, when mice were vaccinated with standard
rabies vaccine and then challenged with street viruses
isolated from fatal cases of human rabies, some
instances of vaccine failure were observed (Wiktor
and Koprowski 1980). Considerable genetic diversity
has also been demonstrated for strains of vesicular
stomatitis virus, using Tl ribonuclease fingerprinting
(Nichol 1988). Using monoclonal antibodies (MAbs),
it has now been shown that variation also exists in
the bovine ephemeral fever (BEF) virus glycoprotein
(Cybinski et al. 1990, 1992).
isolates CSIRO 42 and CSIRO 53 viruses (Standfast
et al. 1984) and the closely related Berrimah virus
(Gard et al. 1983). The Beijing I strain of BEF virus
from China (Zhai et al. 1980) was also compared.
CSIRO 42 virus was isolated from Anopheles
bancroft; mosquitoes collected at Beatrice Hill in the
Northern Territory. CSIRO 53 virus was isolated
from a mixed mosquito pool of Culicine mosquitoes
collected at Etna Creek near Rockhampton in
Queensland. Berrimah virus was isolated from the
blood of a healthy steer located in the north of the
Northern Territory. Neutralisation testing carried out
on this animal for two years revealed high levels of
neutralising antibody to both Berrimah and BEF
viruses prior to the isolation of Berrimah virus
(Cybinski 1987). This indicated a previous infection
with at least one and probably several BEF-related
viruses (Figure I). The Beijing I strain of BEF virus
was isolated in China from bovine blood.
Materials, Methods and Results
Cross-reactivity of REF virus isolates using
polyclonal antibodies
Cross neutralisation tests on the Australian isolates,
using polyclonal antibodies indicated some minor
differences among the BB7721, CSIRO 42 and
CSIRO 53 viruses, while Berrimah virus was con-
sidered to be a different virus, although closely
related (Table I). Testing has not been completed on
the Beijing I virus, although the results so far, using
polyclonal antibodies, indicate that there is little dif-
ference between the Chinese and Australian viruses.
Viruses
The Australian virus isolates which were compared
to the BB7721 strain of BEF virus included the insect
* CSIRO Division of Tropical Animal Production, Private
Bag No.3, Post Office, Indooroopilly, Queensland 4068,
Australia
~1
Paper 3.35
Table 1. Cross-neutralisation titres of Australian BEF virus strains and Berrimah virus, using polyclonal antibodies
BEF Virus
(CSIRO42):Ascitic fluid (BB7721)'
..
BEF BB7721 1024' 512
BEF CSIRO 42 64 384
BEF CSIRO 53 512 1024
Berrimah 1 192 32
Berrimah 2 <2 6
\, ,
, Bovine isolate"
, Mosquito isolate
, Reciprocal of antibody dilution which neutralised 100 TCID,. of virus in 50% of [he wells
(CSIRO53)' (DPP63)'
128
8
1024
32
4
4
<2
32
4096
384
32768
8192
2048
512
128
32
8
2
Isolation Of--.:
Berrimah virus (4.4.81) : t\
--Berrimah virus
-Bovine ephemeral
fever virus
~
0
,fa
'E
"
~~:
I I I I I
20 25
5.11.81
Months
Fig. 1. Antibody titres to BEF and Berrimah viruses found
in serial serum samples from a steer from which Berrimah
virus was isolated.
Strain variation using monoclonal antibodies
Monoclonal antibodies were prepared against the
BB7721 strain of BEF virus and these reacted with
the G, M2 and N proteins according to results
obtained by polyacrylamide gel electrophoresis
(pAGE) and immunoblotting (Tables 2 and 3). Four-
teen of the MAbs against the BEF virus G protein
were neutralising. Indirect immunofluorescence and
neutralisation tests on ascitic fluid from these MAbs
were used to determine their ability to bind and
neutralise the two other Australian strains and
Berrimah virus. The results are listed in Table 2. Only
one MAb, 8D3, failed to neutralise CSIRO 42 virus
while two MAbs, 8B6 and 3D6, failed to neutralise
or bind CSIRO 53. Berrimah virus was neutralised
by only eight of the neutralising MAbs. However,
three additional MAbs were capable of binding to
the virus without neutralisation.
separated by PAGE then transferred to nitro-
cellulose. In Australia, the virus protein preparations
were tested by immunoblotting, for reactivity with
22 MAbs to the G, M2 and N proteins. Results are
shown in Table 4. Four of the G protein-specific
MAbs that bound to the BB7721 strain failed to bind
to the Chinese strain.
Batch variation
To determine what effect, if any, passage level may
have on antigenic variation, three batches of the
BB7721 strain of BEF virus with different passage
histories were tested against 14 neutralising MAbs.
Batch 1 was passaged six times in mouse brain, 34
times in BHK 21 cells, twice in Vero cells then plaque
cloned three times in Vero cells. Batch 2 received the
same number of passages as batch 1 but was plaque
It is thought that those antibodies which bind
without neutralisation may act as blocking antibodies
by binding to the virus and preventing or at least
inhibiting the binding of neutralising antibodies. This
may explain the mechanism by which Berrimah virus
was able to escape neutralisation and cause an
infection in the presence of high levels of neutralis-
ing antibody. Non-neutralising MAbs to the G, M2
and N proteins were tested by indirect immuno-
fluorescence and the results are shown in Table 3.
Of these, two MAbs did not bind to CSIRO 42 virus
while five failed to bind to the CSIRO 53 isolate of
BEF virus. Six non-neutralising G protein Mabs
failed to bind to Berrimah virus.
The Beijing I strain of BEF virus was obtained
in China, and processed by Peter Walker and Helen
Zakrzewski. The virus was purified, the proteins
32
Paper 3.35
Table 2. Viral specificity of neutralising monoclonal antibodies by neutralisation and immunofluorescence to isolates
of BEF virus.
G protein
monoclonal
antibody
Neutralisation titres' of ascitic fluids
(immunofluorescence reaction)
BEF
(BB7721)
BEF
(CSIRO42)
BEF
(CSIRO53)
HER
(DPP63)
512(+)
4096( +)
1024( + )
1024( + )
128(+)
256(+)
256(+)
512(+)
4( +)
512(+)
16(+)
8(+)
8(+)
512(+)
256( +)
2048(+)
3072(+)
2048( +)
384( + )
256( +)
512(+)
256(+)
16(+)
256( +)
6(-)
16(+)
<2( +)
32(+)
256( + )
2048( +)
1024(+)
1024(+)
128(+)
384( + )
512(+)
512(+)
8(+)
<2( -)
<2( -)
8(+)
8(+)
128(+)
<2( -)
<2( +)
<2( -)
<2( +)
96(+)
128(+)
24(+)
128(+)
8(+)
<2(+)
<2( -)
8(+)
8(+)
16(+)
w ~"~~..~~
Table 3. Viral specificity (indirect immunofluorescence) of non-neutralising monoclonal antibodies to isolates of BEF
virus.
Immunofluorescence reactions of hybridoma supernatants
Monoclonal
antibody
1802
3A2
11B5
lOBI
13B5
2C4
18C2
EB4
2C6
F02
4A4
17A3
20A6
11A3
6C1
11B5
205
18A3
1201
901
14B5
14A4
4B4
14A6
12B3
BEF
(BB7721)
BEF
(CSIRO42)--
BEF protein
specificity
BEF
(CSIRO53)
BER
(DPP63)
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
-
+
+
+
+
+
+
+
+
+
+
+
+
+
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
-
+
+
+
+
+
+
+
+
+
+
+
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
-
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
G
G
G
G
G
G
G
G
M2
M2
M2
M2
M2
N
N
N
N
N
N
N
N
N
N
N
N
= binding; -= no binding NT = not tested
133
085
13C6
13A3
1781
9C5
IC6
12A5
1585
1101
306
886
16A6
803
5A5
, Reciprocal of antibody dilution which neutralised 100 TCID,. of virus in 50",0 of the wells
, -' n --' n
Paper 3.35
neutralising MAbs. Protected mice often survived
longer than 14 days compared to 4 or 5 days for the
controls. However. some mice survived no longer
than the controls despite high levels of passive pro-
tecting antibody. indicating the presence of MAb-
resistant virus (Table 7).
cloned separately. Batch 3 was passaged six times in
mouse brain and 25 times in BHK 21 cells then cloned
three times by limiting dilution in Vero cells. Batch
3 was not plaque cloned.
Neutralisation titres for the 14 MAbs against the
three batches of BEF virus are listed in Table 5. Most
MAbs gave the same titres with each batch of virus.
However, batch 1 contained a proportion of virus
which was partially resistant to the MAb 13C6,
shown by virus breakthrough at low dilutions.
Batches 2 and 3 were partially resistant to the MAb
5A5 while batch 2 was also partially resistant to the
MAb 3D6.
Having demonstrated that various batches and
isolates of BEF virus are not homogeneous, variants
were isolated by incubating the BB7721 BEF virus
with 13 of the neutralising MAbs and selecting
plaques which escaped neutralisation. Escape fre-
quencies for these variants, calculated as plaque
forming units/ml in the presence of MAb divided
by plaque forming units in the absence of MAb, are
shown in Table 6. The average escape frequency was
between 10-4 and 10-5, although high escape fre-
quencies were observed for 13C6, 3D6 and 5A5,
while low escape frequencies were obtained for 16A6
and 8B6.
From the BB7721 parent strain of BEF virus, a
total of 43 variants were selected which escaped
neutralisation by the MAb used for selection and in
most cases by other MAbs as well, resulting in 23
patterns of resistance as shown in Figure 2. To deter-
mine whether a variant was resistant or partially
resistant, titres were compared with the titre of the
parent virus. A virus was considered to be resistant
to neutralisation only if the neutralising titre was less
than two. In some cases, there was a greater than
10-fold reduction in the titre, which was considered
to be partial resistance, although viruses showing
only partial resistance to the selecting MAb were
excluded from the study. It was assumed that when
a mutant virus escaped neutralisation by a MAb,
then the epitopes defined by these MAbs were func-
tionally linked. In this way, patterns of neutralisa-
tion and resistance to neutralisation allowed the
variants to be grouped into at least six clusters
representing viruses with mutations affecting the
same antigenic site. Fig. 2. Antigenic map of the BB7721 strain of bovine
ephemeral fever virus. Neutralisation resistant variants were
selected using monoclonal antibodies (MAbs), then tested
for susceptibility (D) or resistance to neutralisation by the
MAb used in selection of the variant (.) and by another
MAb in the panel (*). (.) denotes partial resistance.
Labelling of antigenic sites as 01,02, 03a, 03b is taken
from Cybinski et al. (1990). Antigenic site 04 is a new site
detected by competitive binding assay and by MAb
resistance.
Monoclonal antibody resistance in mice
MAb-resistant mutants were also detected in mice.
Suckling mice inoculated intraperitoneally with
neutralising or non-neutralising MAbs, then
challenged intracerebrally with BEF virus, were
generally protected from paralysis and death by the
134
Paper 3.35
Table 4. A comparison of the Beijing strain I and the Table 5. Neutralisation of BEF virus batches with
BB7721 strain of BEF virus by immunoblot analysis using monoclonal antibodies.
monoclonal antibodies specific for the BB7721 strain.
Monoclonal
antibody
Neutralising titre'
Monoclonal
antibody
Protein
specificity
BB7721 Beijing 1
BEF virus batch number
DB5
13C6
13A3
17BI
9C5
IC6
12A5
15B5
IIDI
3D6
16A6
8D3
5A5
3A2
IlB5
2C4
18C2
EB4
2C6
FD2
20A6
IlA3
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
M2
M2
M2
N
+
+
+
+
+
+
+
+
+
+
+
+
+
:f:
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
j:
+
+
+
+
+
2
DB5
13A3
13C6
17BI
9C5
IC6
15B5
12A5
IIDI
16A6
8D3
3D6
8B6
5A5
256
1024
16
>8192
256
256
256
1024
8
128
256
512
64
256
256
1024
4096
> 8192
256
256
256
1024
8
128
256
16
64
16
256
1024
4096
>8192
256
256
256
1024
8
128
256
512
64
16
, Titres expressed as the reciprocal of the highest dilution required
to neutralise 100 TCID,. of virus in 50"70 of the wells
+ = binding; -= no binding
Escape frequency
Discussion
Previous serological studies using polyclonal anti-
bodies have failed to show any significant differences
between strains of BEF virus from within Australia
or in strains from different countries (Snowdon 1970; Table 6. Frequ:enc,Y with which BEF v,irus. (BB7721)
lnaba 1973; Tian eta!. 1987). However, MAb studies escaped neutrahsatlon by monoclonal antibodies.
on rabies and vesicular stomatitis virus have shown
evidence of extensive antigenic diversity in these Selecting monoclonal antibody
viruses (Wiktor and Koprowski 1980; Luo et al. 1988) and this diversity is thought to be one cause DB5
of vaccine failure for rabies virus. 13A3
l3C6
The data presented here clearly indicate that con- 17Bl
siderable antigenic diversity also exists in BEF virus 9C5
strains from different sources and that variants can lC6
be selected experimentally from cloned BEF virus 15B5
either by passaging or by growing in the presence of l2A5
MAb. The vaccine currently available in Australia ~t~6
is based on the BB7721 strain of BEF virus 3D6
(Vanselow 1985) and this is known to be different 8B6
from viruses now circulating (Cybinski et al. 1992). 5A5
However, the viruses investigated shared the majority
of epitopes as indicated by MAb reactions, therefore
10-4
5xl0-5
6xl0-3
2xl0-5
4xl0-5
3xl0-5
10-4
5xl0-5
5xl0-7
10-5
5xl0-3/10-5.
5xl0-8
10-3
.small plaques/large plaques
135
Paper 3.35
Table 7. Passive protection of mice inoculated with monoclonal antibodies and challenged with BEF virus.
-No deaths recorded
it is not surprising that the vaccine is protective. For
the same reason, it is likely that vaccines against the
BB772 I and the Beijing I strains of BEF virus would
cross protect. On the other hand, there is at least one
virus, Berrimah virus, which has a high degree of
homology with BEF virus strains, but can escape
neutralisation by high levels of BEF antibody.
Berrimah virus is not known to be pathogenic but
other pathogenic viruses could occur which avoid
neutralisation by the same mechanism. These factors
need to be considered when producing an ephemeral
fever vaccine or a diagnostic test based on MAbs.
Monitoring of current field strains of BEF virus is
therefore essential.
Cybinski, D.H., Davis, S.S. and Zakrzewski, H. 1992.
Antigenic variation of the bovine ephemeral fever virus
glycoprotein. Archives of Virology, 124,211-224.
Gard, G.P., Cybinski, D.H. and St George, T.D. 1983.
The isolation in Australia of a new virus related to bovine
ephemeral fever virus. Australian Veterinary Journal, 60,
89.
Inaba, Y. (1973). Bovine ephemeral fever (three-day-
sickness). Stiff sickness. Bulletin Office International
Epizootie, 79, 627-673.
Luo, L., Li, Y., Snyder, R.M. and Wagner, R.R. 1988.
Point mutations in glycoprotein gene of vesicular
stomatitis virus (New Jersey serotype) selected by
resistance to neutralisation by epitope-specific
monoclonal antibodies. Virology, 163, 341-348.
Nichol, S. T. 1988. Genetic diversity of enzootic isolates of
vesicular stomatitis virus New Jersey. Journal of
Virology, 62, 572-579.
Snowdon, W.A. 1970. Bovine ephemeral fever: The
reaction of cattle to different strains of ephemeral fever
virus and the antigenic comparison of two strains of virus.
Australian Veterinary Jo~rnal, 46, 258-266.
Standfast, H.A., St George, T.D. and Dyce, A.L. 1984.
The isolation of ephemeral fever virus from mosquitoes
in Australia. Australian Veterinary Journal, 52, 242.
Tian, F., Jiang, C., Zakrzewski, H. and Davis, S.S. 1987.
A comparison of a Chinese and an Australian strain of
bovine ephemeral fever virus. Australian Veterinary
Journal, 64, 159-160.
References
Cybinski, D.H. 1987. Homologous and heterologous anti-
body reactions in sera from cattle naturally infected with
bovine ephemeral fever group viruses. Veterinary
Microbiology, 13, 1-9.
Cybinski, D.H., Walker, P.J., Byrne, K.A. and
Zakrzewski, H. 1990. Mapping of antigenic sites on the
bovine ephemeral fever virus glycoprotein using
monoclonal antibodies. Journal of General Virology, 71,
2065-2072.
136
Paper 3.35
Wiktor, T.J. and Koprowski, H. 1980. Antigenic variants
of rabies virus. Journal of Experimental Medicine, 152,
99-112.
Zhai, Z., Liu, S. and Pan, W. 1980. A study on the effect
of inoculum dilution on titre and morphological
characteristics of bovine epizootic fever viruses. Acta
Veterinaria et Zootechnica Sinica, II, 201-208.
Vanselow, B., Abetz, I. and Trentfie1d, K. 1985. A bovine
ephemeral fever vaccine incorporating adjuvant Quil A:
A comparative study using adjuvants Qui! A, aluminium
hydroxide gel and dextran sulphate. The Veterinary
Record, 117,37-43.
Wiktor, T.J. and Clarke, H.F. 1973. Comparison of rabies
virus by means of the plaque reduction test. Annales de
Microbiologie (Paris), 124, 283.
137


Antigenic variation in the bovine ephemeral fever virus glycoprotein

http://espace.library.uq.edu.au/view/UQ:290618/TSG_3_35.pdf

Antigenic variation in the bovine ephemeral fever virus glycoprotein

Abstract

Similar works

Full text

Available Versions

University of Queensland eSpace