Journal of General Virology (1990), 71, 2033-2041. Printedin Great Britain
2033
Comparative studies of the proteins of equine herpesviruses 4 and 1 and
asinine herpesvirus 3: antibody response of the natural hosts
Brendan S. Crabb and Michael J. Studdert*
School o f Veterinary Science, The University o f Melbourne, Parkville, Victoria 3052, Australia
Proteins of purified virions of equine herpesvirus 4
(EHV-4; equine rhinopneumonitis), EHV-1 (equine
abortion virus) and asinine herpesvirus 3 (AHV-3) were
compared by metabolic labelling with [35S]methionine
or [14C]glucosamine during growth of low passage
virus in natural host cells (horse or donkey) and high
passage virus in an appropriate cell line and analysis by
SDS-PAGE. Approximately 25 different proteins (Mr
300K to 21 "5K) were clearly resolved for each virus.
The three viruses had similar profiles although significant differences were found. The proteins of the cell
line-grown viruses were similar to their precursor
viruses grown in natural host cells although some small
differences, probably related to differences in glycosylation by the various cell types, were noted. Six or seven
high abundance glycoproteins were identified for EHV4, EHV-1 and AHV-3. The profile of seven glycoproteins of AHV-3 was more similar to EHV-1 than to
EHV-4. Antigenic relationships of the proteins of the
three viruses were examined using radioimmunoprecipitation (RIP) and Western blot analyses and a series of
polyclonal sera raised in colostrum-deprived, specific
pathogen-free (SPF) foals which were immunized with
inactivated EHV-4 (foal 3) or EHV-1 (foal 1),
challenged and cross-challenged; a polyclonal donkey
serum to AHV-3 was also used. The ontogeny of the
antibody response in the SPF foals was studied and the
major immunogenic proteins, as determined by RIP,
were correlated with previously determined serum
neutralizing antibody titres. Antibodies were first
detected 14 days after primary immunization and were
directed to EHV-4 proteins ofM~ 113K, 75K and 56K
or EHV-1 proteins of l l 0 K , 78K, 60K and 58K.
Antibodies to these same three (EHV-4) or four (EHV1) proteins, together with antibodies to the major
capsid protein and proteins of 67K (EHV-4) and 87K
(EHV-1) were detected in response to primary infection (control foal 2) and these sera had high neutralizing antibody titres. The antigens of the three viruses
were extensively cross-reactive with immunodominant
proteins in the Mr ranges 150K to l l 0 K and 62K to
56K. However, cross-absorption of EHV-4 and EHV-1
SPF foal antisera indicated the presence of significant
amounts of type-specific antibody.
Introduction
AHV-3 is more closely related to EHV-1 than is EHV-4
(Browning et al., 1988).
Comparative studies of the proteins of EHV-4 and
EHV-1 reveal similar but not identical overall electrophoretic profiles (Meredith et al., 1989; Turtinen et al.,
1981). Significant and constant differences in electrophoretic mobility of eight of the viral proteins were
recognized when several strains of each virus were
compared (Turtinen et al., 1981). Despite considerable
differences at the genomic level, i.e. distinct restriction
endonuclease DNA fingerprints and low base sequence
similarity, it is clear that EHV-4 and EHV-1 are
genetically collinear and are antigenically related, possessing both shared and unique antigenic determinants
(Studdert et al., 1981; Turtinen et al., 1981; Allen &
Turtinen, 1982; Fitzpatrick & StudderL 1984; Yeargan
et al., 1985; Cullinane et al., 1988). The proteins of
AHV-3 have not been described.
Equine herpesviruses 4 and 1 (EHV-4 and EHV-1) and
asinine herpesvirus 3 (AHV-3) are related alphaherpesviruses infecting members of the family Equidae. EHV-1
(equine abortion virus) is a major cause of abortion and
the recognized cause of abortion 'storms'. EHV-1 is also
considered a cause of respiratory disease and some
strains cause central nervous system disease (Campbell
& Studdert, 1983). EHV-4 (equine rhinopneumonitis) is
a common respiratory pathogen of horses although it has
been occasionally isolated from sporadic abortions
(Sabine et al., 1981 ; Studdert et al., 1981). Both viruses
occur world-wide and are of considerable economic
importance to the equine industry (Allen & Bryans,
1986). AHV-3 was recently described as a respiratory
pathogen of donkeys (Browning et al., 1988). There is
evidence based on serological and DNA analyses that
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2034
B. S. Crabb and M. J. Studdert
The degree of cross-protection between EHV-4 and
EHV-1 remains unclear. Allen & Bryans (1986) described some degree of cross-protection of horses against
EHV-1 challenge after repeated infections with EHV-4.
Using sera from three colostrum-deprived specific
pathogen-free (SPF) foals that were immunized, challenged and cross-challenged with various combinations
of EHV-4 and EHV-1, Fitzpatrick & Studdert (1984)
showed that serum virus-neutralizing antibody responses
were type-specific for foals given EHV-1 but crossreactive after receiving EHV-4. While these results
imply some degree of cross-protection (at least in one
direction) a lack of clinical respiratory disease following
challenge did not allow these investigators to conclude
that adequate protection against EHV-1 challenge had
been achieved by immunization with EHV-4. Natural
infection of horses with EHV-4 does not appear to render
the horse totally immune to EHV-1 infection at least in
providing protection against abortion although a prevalence of respiratory disease has been implicated in
reduced occurrences of abortion (EHV-1) outbreaks
(Doll & Bryans, 1963). These studies suggest that further
work is necessary to define better the antigenic relationships between EHV-4 and EHV-1 particularly at the
level of individual proteins and that their role in
protective immunity requires further study.
The glycoproteins of herpesviruses are present in the
virion envelope and have important functional roles in
infection particularly in mediating entry of the virion
into the host cell via adsorption and penetration. It has
been shown that the immune response to herpes simplex
virus type 1 (HSV-1) is largely directed against the
structural glycoproteins of the virus (Norrild, 1985).
Seven glycoproteins are described for HSV-I: gB, gC,
gD, gE, gG, gH and gI of which gD, gB and gC are highly
immunogenic and appear to be the principal inducers of
virus-specific antibody during HSV-1 infections (Marsden, 1987; Vestergaard, 1980). Allen & Yeargan (1987)
showed that the EHV-1 glycoproteins designated gpl4
and gpl3 were the HSV gB and gC homologues
respectively and proposed, on a basis of genomic
collinearity, that EHV-1 gp17/18 was the homologue of
HSV gE (Alien & Coogle, 1988; Allen & Yeargan, 1987).
EHV-4 and EHV-1 homologues of HSV gB have since
been identified using a gB-specific monoclonal antibody
and shown to be disulphide-linked heterodimers with
polypeptide chains of Mr 76K and 58K (EHV-1) and
74K and 61K (EHV-4) which is similar to the Mr values
for gpl4 and gp17/18 (Meredith et al., 1989). An
uncleaved gB species at 108K (EHV-1)or 112K (EHV-4)
was also detected in low amounts in intact virions but it
was suggested that this protein was not the equivalent of
gpl0.
This study describes comparisons of the structural
proteins of EHV-4, EHV-1 and AHV-3 when low
passage viruses were grown in natural host cells (horse or
donkey) and after adaptation and passage of the viruses
in heterologous cell lines. The major glycoproteins of
each virus were identified. Proteins of the three viruses
were further compared by identifying the major immunodominant proteins (or regions of immunodominance) of each virus in the natural hosts and by examining
the extent of antigenic cross-reactivity using radioimmunoprecipitation (RIP) and Western blotting and
panels of SPF foal sera prepared by Fitzpatrick &
Studdert (1984) and a donkey serum to AHV-3 (Browning et al., 1988). The ontogeny of the antibody response
to EHV-4 and EHV-1 in the SPF foals was investigated
using RIP analysis and these results were correlated with
the serum neutralizing antibody titres determined by
Fitzpatrick & Studdert (1984).
Methods
Cells and viruses. Virus strains 405/76 and 438/77 were used as
representatives of EHV-4 and EHV-1 respectively. These viruses were
grown in equine foetal kidney (EFK) cells and used at passage 11
(EHV-4) and 18 (EHV-1) as described elsewhere (Studdert, 1983;
Studdert & Blackney, 1979). An AHV-3 isolate, designated strain
804/87 (Browning et al., 1988), was used after four passages in donkey
foetal kidney (DFK) cells. Each virus strain was adapted to growth in a
heterologous cell line. Monolayer cell cultures used in the adaptation
and passaging of virus were infected initially with approximately 0.1
TCIDso per cell and in later passages with I to 5 TCIDs0 per cell. EHV1 and AHV-3 were grown in the same cell line and used at passage 45
and 11 respectively. EHV-4 was adapted to a different cell line and used
at passage 65. Both cell lines were grown in M E M (Gibco-BRL)
containing Earle's salts, L-glutamine and non-essential amino acids and
supplemented with 5% foetal bovine serum (Gibco-BRL) and 0-08 MNaHCO3. Infected cells were maintained in M E M supplemented with
1% foetal bovine serum, 40 ~tg/ml gentamicin, 4 ~tg/ml trimethaprim,
0.13 M-NaHCO 3 and 0-015 M-HEPES (Sigma) at pH 7.4.
Infection and radiolabelling. Monolayer cell cultures were infected in
25 cm 2 cell culture flasks with 5 to 20 TCIDso per cell. After adsorption
for 1 h at 37 °C the inoculum was removed and replaced with
maintenance medium containing one-fifth the normal concentration of
methionine. At 6 h post-infection (p.i.) 5 ~tCi/ml of [35S]methionine
(Amersham) was added to each flask. For labelling of glycoproteins,
infected cell cultures were maintained in maintenance medium until 5 h
p.i. at which time the medium was replaced with maintenance medium
containing half the normal concentration of glucose and 1 p,Ci/ml
[~4C]glucosamine (Amersham). At 22 to 36 h p.i. virus was purified
from the cell culture medium (see below). Infected cell proteins were
prepared for RIP by scraping the cells into the cell culture medium and
centrifuging the virus/infected cell mixture at 100000 g for 1 h (SW60
rotor). The resulting pellet was disrupted in RIP lysis buffer (0.05 MTris-HC1 pH 7.5, 2% Triton X-100, 0.6 M-KC1), sonicated briefly (1 to 2
s) at a low setting and left on ice for 30 rain. The disrupted pellet was
centrifuged again at 100000g for 1 h at 4 °C and the supernatant used in
immunoprecipitations.
Viruspurifieation. Purification of virus from infected cell culture fluid
was based on the method of Schrag et al. (1989). Briefly, the medium
was clarified at 500 g for 15 rain followed by a second clarification at
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Proteins o f EHV-4, E H V - 1 and A H V - 3
2500 g for 15 min. Extracellular virus was then pelleted at 50000 g for
90 min, resuspended in a small volume of 0-01 M-Tris-HCI pH 7-4, 0.1
M-NaCI and 0-00I M-EDTA (TNE) buffer and layered onto preformed
5 to 15% Ficoll-400 (Pharmacia) gradients in TNE and centrifuged at
20000 g for 2 h at 4 °C. The gradients were fractionated from the
bottom and assayed for radioactivity. Peak fractions were diluted
fivefold in TNE, pelleted again, resuspended in a small volume of TNE
(20 ~1) and stored at - 2 0 °C.
Antisera. Fitzpatrick & Studdert (1984) carried out experiments in
which three colostrum-deprived SPF foals were immunized intramuscularly with inactivated EHV-1, cell lysate or inactivated EHV-4 (foals
1, 2 and 3 respectively) and challenged and cross-challenged with
combinations of EHV-1 and EHV-4. Procedures for preparing and
inactivating inocula and for immunizing the foals were described
previously (Fitzpatrick & Studdert, 1984); the immunization and
challenge protocols are also incorporated in Fig. 3. Sera were obtained
twice weekly from all foals over the time-course of the experiment
(approx. 120 days) and serum neutralizing antibody titres determined
for each serum. For comparison these data are incorporated in Fig. 3.
For the further ontogeny studies carried out in this study a selection of
the SPF foal sera were used in RIP experiments. For Western blot and
absorption studies selected sera used were those taken just prior to
cross-challenge, i.e. at day 77 for foal I and at day 60 for foal 3.
Antiserum to AHV-3 was obtained at 40 days after experimental
infection of a seronegative, weanling donkey with nasal washings from
a second donkey that had received high doses of corticosteroids that
reactivated latent AHV-3 (Browning et al., 1988).
Radioimmunoprecipitations. Protein G Sepharose 4 Fast Flow
(Pharmacia) beads (PG) were prepared for RIP by washing three times
in 0.05 M-Tris HC1 pH 8.0 containing 1 mM-EDTA, 0-15 M-NaCI,
0.25% bovine serum albumin (BSA) and 1% Triton X-100 (Sigma, RIP
buffer). Antigen was precleared by incubating day 0 SPF foal sera with
radiolabelled antigen (25 ~tl serum per ml of solubilized antigen) for 1 h
on ice. The antigen was then used to twice resuspend a washed pellet of
PG beads (25 ~tl PG packed beads per ml antigen) incubating the
mixture each time for 30 rain on ice. The beads and any non-specifically
bound material were removed by brief centrifugation at 12000 g for 5 s.
Aliquots of precleared antigen (approx. 50000 c.p.m.) were incubated
with 5 ~tl of PG beads that bad been previously saturated with 50 ~tl of
1/10 dilution of antiserum. The mixture was shaken for 30 rain at 4 °C
after which time the beads were washed twice in RIP buffer followed
by a further wash in the absence of BSA. The beads were then
resuspended in Laemmli sample buffer comprising 2% (w/v) SDS, 5 %
(v/v) 2-mercaptoethanol, 10% (v/v) glycerol in 0.5 M-Tris HC1 pH 6.8
and the immune complexes were disassociated by boiling for 3 min.
SD~PAGE. This was carried out according to Laemmli (1970).
Briefly, electrophoresis was performed on 10% polyacrylamide slab
gels (16 cm × 16 cm × 0.75 mm) cross-linked with bisacrylamide and
with reservoir and sample buffers containing 0.1% and 2% SDS
respectively. Prior to electrophoresis all protein samples were boiled for
3 rain in Laemmli sample buffer. A mixture of ~C-methylated Mr
standards (Amersham) were used in each electrophoresis run and
contained myosin (200K), phosphorylase b (92.5K), bovine serum
albumin (69K), ovalbumin (46K), carbonic anhydrase (30K) and
trypsin inhibitor (21-5K). After electrophoresis proteins were fixed in
7-5% acetic acid and prepared for fluorography by soaking the gel in
fluorographic reagent (Amplify, Amersham) for 30 min. The dried gels
were exposed to X-ray film (Kodak, X-Omat AR) at - 7 0 °C for 2 to 6
days.
Western blots. Approximately 50 gg of purified virus was run on 10%
polyacrylamide gels and the separated proteins were transferred to
Immobilon PVDF Transfer Membranes (Millipore) using the Bio-Rad
Trans-Blot system in a transfer buffer containing 20% methanol, 50
2035
mM-Tris-HCl, 380 mM-glycine and 0.1% SDS at 30V for 18 h. The gel
and the membrane were equilibrated in transfer buffer for 15 min prior
to transfer. After transfer unoccupied sites on the membrane were
blocked by soaking in 20% normal goat serum in phosphate-buffered
saline containing 0.3 % Tween-20 (PBST) for 2 h at room temperature
(RT). Membranes were then probed with virus-specific antibody
diluted 1/50 in PBST using a multi-chanelled antibody probing
apparatus (Immunetics) for 1 h at RT. The blots were washed four
times with PBST and soaked in horseradish peroxidase-conjugated
goat anti-horse antibody (Kirkegaard and Perry Laboratories) diluted
1/200 in PBST, for a further 1 h at RT. Blots were washed again and
developed using a tetramethylbenzidine (TMB) substrate system
(Kirkegaard and Perry Laboratories). The identity of glycoproteins was
independently confirmed using a Glycan Detection Kit (Boehringer
Mannheim) which is based on the method of O'Shannessy et al. (1987).
Absorption ofSPFfoal sera. Confluent monolayers of the appropriate
cell line in 890 cm 2 roller bottles were infected with EHV-4 or EHV-1
respectively. At 18 h p.i. infected cells were scraped into the medium,
washed twice in cold PBS, divided into tubes containing approximately
2 x 107 cells per tube and stored at 4 °C. Five-hundred lal of SPF foal 1
or 3 serum, diluted 1/10 in PBS, was used to resuspend cells infected
with heterologous virus. Suspensions were incubated with intermittent
vortexing for 1 h at 37 °C. The tubes were then centrifuged and the
supernatant was removed and added to the next tube completing one
absorption step. The sera were tested for reactive antibody in RIP
analysis after four and six absorption steps.
Results
Comparisons o f the structural proteins o f EHV-4, E H V - 1
and A H V - 3
Virions of EHV-1, EHV-4 and AHV-3 were radiol a b e l l e d w i t h [ 3 s S ] m e t h i o n i n e (Fig. 1 a n d 2) or w i t h
[ 1 4 C ] g l u c o s a m i n e (Fig. 2), p u r i f i e d o n 5 to 15% Fico11400 g r a d i e n t s a n d a n a l y s e d b y S D S - P A G E . F i g . l s h o w s
f l u o r o g r a p h i c i m a g e s o f t h e p r o t e i n profiles o f t h e t h r e e
v i r u s e s a n d e x a m i n e s c h a n g e s t h a t o c c u r in t h e s e profiles
w h e n l o w p a s s a g e viruses, g r o w n in n a t u r a l h o s t cells
( E F K cells for E H V - 4 a n d E H V - 1 o r D F K cells for
A H V - 3 ) , w e r e a d a p t e d to g r o w t h in cell lines. O v e r a l l t h e
p r o t e i n profiles o f E H V - 4 a n d E H V - 1 in F i g . 1 are v e r y
s i m i l a r to t h o s e p r e v i o u s l y r e p o r t e d for o t h e r s t r a i n s o f
t h e s e t w o v i r u s e s ( A l l e n & B r y a n s , 1986; M e r e d i t h et al.,
1989; T u r t i n e n et al., 1981). A p p r o x i m a t e l y 25 d i f f e r e n t
p o l y p e p t i d e s in a Mr r a n g e o f 3 0 0 K to 2 1 . 5 K w e r e c l e a r l y
r e s o l v e d for e a c h virus. T h e profile o f A H V - 3 is v e r y
s i m i l a r to t h o s e o f b o t h E H V - 4 a n d E H V - 1 .
P r o t e i n profiles s h o w i n g s t r u c t u r a l p r o t e i n s f r o m l o w
passage EFK/DFK-grown
v i r u s e s are v e r y s i m i l a r to
t h o s e o f the s a m e v i r u s a f t e r a d a p t a t i o n to a cell line.
S o m e s m a l l d i f f e r e n c e s are a p p a r e n t h o w e v e r a n d t h e s e
usually a p p e a r as s m a l l M r shifts s u c h as in t h e 9 2 . 5 K to
6 9 K r e g i o n s o f A H V - 3 . A 6 7 K E H V - 4 p r o t e i n ( s h o w n to
be a g l y c o p r o t e i n , Fig. 2) a p p e a r s in t h e profile o f t h e cell
l i n e - g r o w n virus. T h e cell l i n e - g r o w n v i r u s e s w e r e u s e d in
all s u b s e q u e n t e x p e r i m e n t s .
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2036
B. S. Crabb and M. J. Studdert
Mr
E4
E F K CL 1
E1
E F K CL2
A3
DFKCL2
3s S
~
E4
1. C
e~
A3
El
35S
3s S
14C
14C
270K
200K
MCP
~:. . . . .
ll0K
x
92-5K
:::5:i
.~:
77K
--/~75K
69K
,-62K
46K
i;
--56K
--87K
--78K
? 3-1:i:
.... ~ 6 0 K
i~~! --58K
$~'
! ! ~i~ii~ ~
~60K
--57K
:--49K
,. : ~ 4 4 K
i
30K
..i:i~::i ¸¸ i•• )7.
i: ~ -~7
"
21.5K t
Fig. 1. S D S - P A G E of the vmon proteins of purified EHV-4 (E4),
EHV-1 (El) and AHV-3 (A3). Proteins were labelled metabolically
with [35S]methionine from 6 to 24 h p.i. The profiles of the three viruses
when grown in low passage, natural host cells (EFK or D F K ) or an
appropriate cell line (CL1 for EHV-4 and CL2 for EHV-1 and AHV-3)
were compared. Viruses were purified on 5 to 15 ~ Ficoll 400 gradients
and analysed by SDS P A G E under reducing conditions. All samples
were run on the same 10~ polyacrylamide gel and photographed from a
single fluorographic image. The position of the major capsid protein
(MCP, Mr 150K) is indicated. The Mr standards used are myosin
(200K), phosphorylase b (92.5K), bovine serum albumin (69K),
ovalbumin (46K), carbonic anhydrase (30K) and trypsin inhibitor
(21.5K).
Fig. 2 shows [~4C]glucosamine-labelled glycoproteins
of the three purified cell line-grown viruses next to a
corresponding [3SS]methionine-labelled virion profile.
The EHV-1 profile shows glycoproteins of Mr 250K,
ll0K, 87K, 78K, 60K, 58K and 49K which are in good
agreement with those previously published for EHV-1
strain A 183 (Allen & Yeargan, 1987; Turtinen, 1983) and
strain Abl (Meredith et al., 1989) and appear to
correspond to the six high abundance glycoproteins
described by Allen & Yeargan (1987) and Allen et al.
(1987), namely gp2, gpl0, gpl3, gpl4, gp17/18 and
gp21/22a. The EHV-4 glycoprotein profile shows major
glycoproteins of Mr 270K, 113K, 77K, 75K, 67K, 62K
and 56K. The glycoprotein profile of AHV-3 shows
similarities to the EHV-1 profile with proteins of Mr
250K, 150K, 115K, 82K, 78K, 60K and 57K. A second
method for detecting glycoproteins involving electrophoretic separation and immobilization of viral proteins
on PVDF membranes (Glycan Detection Kit, Boehringer Mannheim) confirmed the above results as well as
allowing better resolution of the 77K/75K bands of
Fig. 2. S D S - P A G E o f t h e virionproteins and glycoproteins of purified
cell line-grown viruses EHV-4 (E4), EHV-1 (El) and AHV-3 (A3)
labelled with psS]methionine and [l*C]glucosamine respectively.
Labelling, purification and electrophoresis were as described in Fig. 1
except that [14C]glucosamine was added from 5 to 24 h p.i. All samples
were run on the same 10~ polyacrylamide gel. The Mr values of the
major glycoproteins, indicated to the right of each [~*C]glucosamine
lane, are derived from Mr standards as in Fig. 1.
EHV-4 and detecting a AHV-3 glycoprotein of 44K (data
not shown).
Ontogeny of the antibody response in S P F foals
Antisera were obtained from three colostrum-deprived,
SPF foals following a series of immunizations, challenges
and cross-challenges with combinations of EHV-4 and
EHV-1. These sera were previously tested for serum
neutralizing antibodies (Fitzpatrick & Studdert, 1984)
and the results for each foal are shown in Fig. 3 (a) (foal
i), Fig. 3(b) (foal 2) and Fig. 3(c) (foal 3). A selection of
these sera were used to immunoprecipitate [3SS]methio n_
ine-labelled EHV-4 or EHV-1 proteins to investigate the
ontogeny of the antibody response in SPF foals to
individual viral proteins.
None of the foals had EHV antibody before primary
immunization (foals 1 and 3) or experimental infection
(foal 2). A low antibody response was observed for both
foals 1 and 3 (Fig. 3a and 3e) 14 days after primary
immunization with inactivated EHV-1 and EHV-4
respectively. Three (foal 3) or four (foal 1) immunoreac-
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Proteins of EHV-4, EHV-1 and AHV°3
(a)
(c)
(b)
Foal 1
Days
Foal 2
i
|
J
I1
C1
C1
C4
Days
r
i
E4 0 14 38 60 81,107E10 14 38 60 81 107
-I10K
I1
Foal 3
Days
Days
I
E1 0 6 14 2 8 3 8 4 9 6 0 7 0 81 91107118
....
E4 0 7 14 28 35,49 63 77 87 98
J
15K
--78K
-75K
-60K
"58K
-56K
C4
~' 4 + 4 '~
IL ILC1 C1C4
4 4 4 4 '~
IL IL C1C1C4
I4
I4
i
lOO
...............
20
40
60
_" __
80
Time p.i. (days)
200
100
2037
120
~
,
,
,
C4
i
C1
!
C1
.k
/--
I
]
50
i
00 20 40 60- 80 I 120
100
Time p.i. (days)
z 0.0
20
40
60
80
Time p.i. (days)
100
Fig. 3. Immunoprecipitation of [35S]methionine-labelled, detergent-solubilized preparations of EHV-4 (E4) and EHV-1 (El) by sera
from three colostrum-deprived, SPF foals that were immunized with inactivated EHV vaccines, challenged and cross-challenged w i t h
EHV-4 and EHV-1. The days of immunization (I) or challenge (C) with either EHV-4 or EHV-1 (1) or immunization (foal 2) with mockinfected cell lysate (L) are indicated by the arrows and the appropriate code (I1, I4, C1, C4, or IL) immediately beneath each gel. Foals I
(a) and 3 (c) sera were used to immunoprecipitate proteins of the virus with which they were immunized, i.e. EHV-1 for foal 1 and EHV4 for foal 3; foal 2 (b) sera were used to immunoprecipitate the proteins of both EHV-4 (left side) and EHV-1 (right side). Proteins of
[35S]methionine-labelled purified virus are shown in the left-hand lane of each serum set. Mr values of some of the major
immunodominant proteins are indicated to the right of gels (a) ~md (c). Serum neutralizing antibody titres (SN Ab titre) to EHV-4 (
)
and EHV-1 (-...) for all sera, derived from earlier studies (Fitzpatrick & Studdert, 1984), are reproduced beneath each gel. The SN Ab
titre is the reciprocal of the highest dilution of each serum that neutralized approximately 100 TCID5o.
tive proteins were observed at this time with Mr values
corresponding to apparently comparable EHV-4 and
EHV-1 proteins of 113K/110K, 75K/78K and 56K/60 to
58K. Sera from foals 1 and 3 precipitated considerably
more viral antigens after secondary immunizations (day
28 onwards). Maximal antibody responses in foals 1 and
3 were recognized after challenge with live, homologous
virus as seen by the increase in intensity of the bands at
day 49. This did not correspond to maximum serum
neutralizing antibody titres. Although the intensity of
bands generally decreased in foals 1 and 3 for sera tested
after day 49, the amount of neutralizing antibody either
increased (foal 1) or remained relatively constant (foal 3).
Cross-challenge did not result in antibodies being
produced that were capable of precipitating any new
viral antigens in either foal I or 3; however, the increase
in intensity of the bands at day 118 for foal 1 and day 98
for foal 3 shows an increase in antibody response after
the second cross-challenge in both foals. The control, foal
2, had detectable antibody after primary infection with
live EHV-1 (day 60) (Fig. 3b). The same three or four
immunoreactive EHV-4 and EHV-1 proteins as described above were detected by these sera. The antibody
response of foal 2 was boosted significantly after EHV-4
challenge as seen by the increase in the number and
intensity of EHV-4 and EHV-1 proteins on day 107. The
serum neutralizing antibody titres of the control foal 2
first indicated the presence of EHV-1 or EHV-4 antibody
after day 64 or day 88 respectively; however, the
antibody titres Of foal 2 sera were very high, especially
against EHV-1, after about day 95, although only a
relatively small number of immunoreactive proteins of
low intensity were detectable by RIP at day 107.
Immunogenieity and cross-reactivity of EHV-4, EHV-1
and AHVo3 antigens
Fig. 4 and 5 show the extent of cross-reactivity of EHV-1,
EHV-4 and AHV-3 antigens using selected polyclonal
sera to each virus. Fig. 4 shows Western blots of purified
viral preparations of EHV-1, EHV-4 and AHV-3 probed
with each of the three sera. No reactivity was seen when
day 0 sera were used to probe these Western blots.
Antibody to eight to 12 structural protiens of each virus
was detected and there was extensive cross-reactivity
between the three viruses. Comparisons of the Mr values
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2038
(a)
MCP
l13K--
B. S. Crabb and M. J. Studdert
Post
Pre
EEAEEA
41341
~
(b)
Post Pre
EE AE EA
41 34 1 3
MCP . . . .
110K~
64K . . . .
62K-- ~ ~:
56K~
58K-
,~
~;'.;-rg-
(c)
Post
Pre
E EAEEA
4 1 3 4 1 3
MCP
115K ......
'
~
57K-- ~
O
331{-
33K-
33K--~
labelled EHV-4, EHV-1 and AHV-3 antigens. There was
a progressive reduction in cross-reactive antibody in the
EHV-4 and -1 antisera from absoption steps 1 to 6. By six
absorptions all cross-reactive antibody was removed as
indicated by the failure of these absorbed antisera to
immunoprecipitate proteins of the virus used for their
absorption. The remaining antibody, reactive with
proteins from the virus against which it was originally
raised, is considered type-specific for that virus. The
detection of all the structural proteins eliciting typespecific antibody is difficult, however; EHV-4 and
EHV-1 proteins corresponding to gpl0 (113K and 110K
respectively) and EHV-1 proteins corresponding to
gp17/18 (60K/58K) are precipitated by their respective
absorbed sera. Some AHV-3 antigens are precipitated by
both absorbed sera.
Discussion
Fig. 4. Western blot analysis of the proteins of EHV-4 (a), EHV-1 (b)
and AHV-3 (c) using selected pre- and post-EHV-4 (E4) and EHV-1
(El) SPF foal sera and a pre- and post-infection AHV-3 (A3) donkey
serum. Viral proteins prepared from purified virions were separated on
10% polyacrylamide gels and transferred to PVDF membranes.
Membranes were incubated with primary antibody diluted 1/50 in a
multi-chanelled antibody probing apparatus followed by incubation
with horseradish peroxidase-conjugated goat anti-horse antibody
diluted 1/200 and developed using a TMB substrate. Mr values of the
immunodominant structural proteins and the MCP are shown to the
left of each panel.
from SDS-PAGE of [14C]glucosamine-labelled virus
proteins show that antibody was present to proteins
having Mr values comparable to those of the major
glycoproteins of each virus. Some proteins appear to be
significantly more immunodominant than others, i.e.
l l 3 K / l l 0 K / l l 5 K , 62K/60K/60K and 56K/58K/57K
proteins of EHV-4, EHV-1 and AHV-3 respectively.
Other non-glycosylated proteins such as a 150K protein,
probably the major capsid protein (MCP), are also
strongly immunogenic. The EHV-1 serum possesses
antibodies to a 64K EHV-1 protein that does not react
with either EHV-4 or AHV-3 sera; there was also a 33K
EHV-1 protein that reacted with EHV-1 and AHV-3 sera
but not with EHV-4 serum. Otherwise differences in
reactivity between the sera and a particular protein were
usually seen as variations in intensity rather than the
absolute presence or absence of a band.
A method of cross-absorbing EHV-4 and EHV-1 SPF
foal sera with cells infected with heterologous virus was
developed to investigate the relative amounts of typespecific antibody present in the sera. EHV-4 (Fig. 5a)
and EHV-1 (Fig. 5b) antisera were tested, both before
absorption and following four and six absorption steps,
for their ability to immunoprecipitate [3SS]methionine-
Our desire to work with high titre, cell line-grown viruses
led us to compare the protein profiles of these viruses
with those of the precursor viruses grown at low passage
in natural host cells and to examine the possibility of
significant changes occurring as a result of the adaptation process. The number of passages in the cell lines, 65
for EHV-4, 45 for EHV-1 and 11 for AHV-3, were low
when compared with EHV-1 strains KyA-ha or KyALM which were adapted from the KyA foetal strain and
passaged extensively in Syrian hamsters or mouse L-M
cells respectively over a period of more than 10 years
(Perdue et al., 1974). O'Callaghan & Randall (1976)
reported only slight differences in the protein profiles
between these extensively passaged host range mutant
strains, primarily in some minor envelope glycoproteins.
Our findings show small differences in the profiles after
adaptation and somewhat limited passage. The proposal
by Turtinen et al. (1981) that different cell types may
glycosylate viral proteins differently because they may
possess distinct glycosyl transferases would explain the
small shifts in Mr exhibited by some viral proteins after
adaptation to cell lines.
The total protein profiles for the three viruses are very
similar and they are obviously closely related herpesviruses. The glycoprotein profile of EHV-4, however,
was clearly different to those of EHV-1 and AHV-3.
There are two main possibilities to explain this: (i) the
changes are the result of differences at the level of DNA
that result in carbohydrate modifications that are
specific for a particular virus type; (ii) the cell lines used
differently glycosylate protein backbones that are more
similar than the relatively large migrational differences
exhibited by some glycoproteins might suggest. The first
point implies that AHV-3 is indeed closer in evolutionary
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Proteins o f EHV-4, E H V - 1 and A H V - 3
(a)
Unabsorbed
i'
Mr
E4
11
El
A3
(b)
EHV-4 antiserum
x 4 abs steps
× 6 abs steps
l I
E4
E1
A3
E4
II
E1
A3
EHV-I antiserum
× 4 abs steps
Unabsorbed
I
E4
E1
A3
I
I
E4
El
A3
x 6 abs steps
I
I
E4
E1
A3
;~
~
--gpl0
~
.....
•
J
";:3;:;
•:•;••~i,¸ ,~•,;•~;~
2039
:~..........
":
- - g p 17/18
•;•
t
Fig. 5. Immunoprecipitations of [3sS]methionine-labelled EHV-4 (E4), EHV-1 (El) and AHV-3 (A3) proteins using an EHV-4
antiserum (a) or an EHV-1 antiserum (b) from SPF foals. Each serum was used to immunoprecipitate proteins of each of the three
viruses either unabsorbed or after four or six absorption steps ( × 4 or × 6 abs steps) EHV-4 antiserum was absorbed with EHV-1infected cells and EHV-1 antiserum was absorbed with EHV-4-infected cells. The positions of EHV-1 glycoproteins gp 10 (11 OK) and
gp17/18 (60K/58K) are indicated. The MT standards are as in Fig. 1.
distance to EHV-1 than is EHV-4 as previously
suggested by Browning et al. (1988). However, the slight
difference in the profiles of EFK-grown and cell linegrown EHV-4, such as the appearance of a glycoprotein
at 67K, and the fact that EHV-1 and AHV-3 were both
adapted to growth in the same cell line, suggests that the
second point may at least be a contributing factor to the
observed differences.
Although the glycoprotein profile of EHV-1 (and
AHV-3) was very similar to those reported for other
EHV-1 strains the EHV-4 profile possesses both similarities and differences to the MD strain of EHV-4 described
by Meredith et al. (1989). The most notable differences
are the absence of a detectable glycoprotein in the 92K
region and the presence of 77K and 67K glycoproteins in
the cell line-grown EHV4.405/76 used in this study. The
92K protein of the EHV4. MD is heavily glycosylated (as
seen by the amounts of [14C]glucosamine incorporated)
in comparison to other glycoproteins whereas the EHV-4
strain used in the present study does not have any such
dominant glycoproteins. It is possible, therefore, that the
gp92 equivalent is less glycosylated in this particular
strain and therefore appears as a protein of lower Mr i.e.
at 77K or 67K. Monoclonal antibodies are required to
identify definitively the structurally homologous
proteins.
In the study of the ontogeny of the antibody responses
to EHV-4 and EHV-1 in the SPF foals, antibodies to
viral proteins were first detected at day 14 after primary
immunization with either EHV-1 or EHV-4. This initial
antibody response was directed to four (EHV-1) or three
(EHV-4) proteins in foals 1 and 3 respectively that
correspond to EHV-1 proteins of l l 0 K , 78K, 60K and
58K and EHV-4 proteins of 113K, 75K and 56K all of
which are likely to be glycoproteins. A minor nonglycosylated protein has been shown to have an Mr
similar to the 110K EHV-1 glycoprotein and although
unlikely it cannot be discounted as the major immunogen
seen at l l 3 K / l l 0 K for EHV-4/EHV-1 respectively
(Turtinen, 1983). The four EHV-1 proteins have Mr
values corresponding to gpl0, gpl4 and gp17/18 of Allen
& Yeargan (1987). It is possible that these are not all
different species but are some of the three Mr forms of
the gB homologue described by Meredith et al. (1989)
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2040
B. S. Crabb and M . J. Studdert
and subsequently by Sullivan et al. (1989). This is as yet
unclear as the latter authors did not attempt to reconcile
the earlier view of Allen & Yeargan (1987) that gp17/18
maps to the Us component of the EHV-1 genome despite
the smaller component of gB having a similar Mr to
gp17/18, a view incompatible with the single gene
location of gB in UL. One must assume that they
subscribe to the suggestion by Meredith et al. (1989) that
two comigrating but distinct glycoproteins are present.
Nevertheless, gpl4 (the large component of the HSV gB
homologue) appears important in the early antibody
response to EHV immunization.
The control foal 2, which received mock-infected cell
lysate and was experimentally infected at day 38 with
EHV-1, produced antibodies that were directed to the
same three or four proteins (probably glycoproteins) as
observed in the post-inactivated virus vaccine responses
of foals 3 and 1. Serum from foal 2 after cross-challenge
with EHV-4 precipitated additional EHV-1 proteins of
150K and 87K which correspond to the MCP and gpl3
(the HS¥ gC homologue) and EHV-4 proteins of 150K
(MCP) and 67K which may be a lower Mr form ofgpl3.
The serum neutralizing antibody titres of foal 2 sera after
EHV-4 cross-challenge were high, especially against
EHV-1 (day 107). It would appear that some or all of the
MCP and EHV-1 glycoproteins corresponding to gpl0,
gpl3, gpl4 and gp17/18 and their EHV-4 counterparts
are important immunogens in the naturally infected host,
eliciting antibody in response to live virus that is strongly
neutralizing. It would be of interest, particularly in the
context of subunit vaccine development, to determine
the relative immunological importance of these glycoproteins. Further identification of EHV-4 and EHV-1
glycoproteins structurally homologous to glycoproteins
of HSV or other alphaherpesviruses would be useful for
this process.
In the case of foals 1 and 3, immunized with
inactivated EHV-1 and EHV-4 respectively, a maximal
antibody response was detected by RIP following first
challenge with live virus (day 49) that was significantly
stronger, in terms of the number and intensity of
precipitating proteins, than the antibody response
observed at any time for foal 2. The serum neutralizing
antibody titres were not high at this time. In short,
antibody produced in foal 2 in response to experimental
infection with live (and presumably replicating) virus is
far more potent neutralizing antibody than that produced in foals 1 and 3 following immunization with
inactivated virus, despite the fact that much more
antibody to a wider range of proteins is produced in the
latter as detected by RIP. It has been proposed that the
biological significance of a large spectrum of antibodies
to HSV proteins is in the formation of immune
complexes resulting in elimination by uptake by macro-
phages (Norrild, 1985). This may account for the high
levels and more diverse nature of the antibodies observed
in foals 1 and 3 whose immune system would regard
inactivated virus as merely foreign antigen. It appears
from the present study that following infection with live
virus a population of B lymphocytes is selected that
produce antibodies directed to epitopes relevant in
neutralization as is necessary to limit effectively the
spread of virus and eliminate infectious virus from the
host.
The Western blots showed that the immunogenic
antigens of each virus are largely cross-reactive. The
immunodominant viral proteins are found in the region
of 150K to 11OK and 62K to 56K and it is likely that the
proteins include the MCP, gpl0 and gp17/18. Proteins
having Mr values comparable to all the major glycoproteins were shown to elicit some antibody (although
this is not clear from the Western blot chosen for Fig. 4)
as were several other non-glycosylated proteins. Absorption experiments (Fig. 5) revealed the presence of typespecific epitopes on several infected cell/viral proteins
including the immunodominant proteins that correspond
to gp 10 and gp 17/18. Though non-quantitative it appears
that as many as 20~ to 50~ of the epitopes of both EHV4 and EHV-1 are type-specific. The degree of typespecificity presumably depends on the conserved nature
of a particular protein; i.e. between EHV-4 and EHV-1,
gpl4 is thought to be highly conserved whereas gpl3
possesses predominantly type-specific epitopes (Sullivan
et al., 1989; Allen & Coogle, 1988). Our findings echo
those of Yeargan et al. (1985) who found that about 30~
of monoclonal antibodies to EHV-4 and EHV-1 (termed
subtypes 2 and 1 respectively) were type-specific. It is
likely that most (all) of the glycoproteins possess typespecific epitopes, but the technique of RIP was not a
sufficiently sensitive immunological detection method to
detect antibody to these proteins.
Analyses of the structural proteins/glycoproteins of
AHV-3 reveal remarkable similarities to both EHV-4
and, especially, EHV-1. The extensive antigenic crossreactivity between the three viruses shows that AHV-3
shares a large number of epitopes with both equine
viruses although epitope differences are highlighted in
the absorption experiments by the ability of both EHV-1and EHV-4-absorbed sera to immunoprecipitate some
AHV-3 antigens (Fig. 5). These findings lend support to
those of Browning et al. (1988) who suggested that AHV3 is more closely related to EHV-1 than EHV-4 and focus
attention on the origin and natural history of EHV-1
infection in the horse.
Wethank NinoFicorillifor excellenttechnicalassistance.Financial
supportwasprovidedby MelbourneUniversityEquineResearchFund
and Ministryof Sport and Recreation, Victoria.
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Proteins of EHV-4, EHV-1 and AHV-3
Note added in proof Since preparing the manuscript
we have obtained monoclonal antibodies specific for the
high abundance glycoproteins of EHV-4 and EHV-1
from Dr G. P. Allen, University of Kentucky, Lexington, U.S.A. Using Western blot and radioimmunoprecipitation analyses the identity of the structural glycoproteins and immunoreactive proteins as designated in our
paper was confirmed. Individual monoclonal antibodies
specifically identified gp2, gpl0, gpl3, gpl4, gp17/18
and gp21/22 as described for EHV-1 by Allen & Yeargan
(1987). EHV-4 gp67 was confirmed as a low Mr form of
gpl3, a possibility discussed in our paper.
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