Immunization of BALB/c mice with DNA encoding equine herpesvirus 1 (EHV-1) glycoprotein D affords...

Immunization of BALB/c mice with DNA encodingequine herpesvirus 1 (EHV-1) glycoprotein

D affords partial protection in a modelof EHV-1-induced abortion

Catherine Walkera, Kirsten M. Ruitenberga,Daria N. Loveb, J. Millar Whalleya,*

aDepartment of Biological Sciences, Division of Environmental and Life Sciences,

Macquarie University, Sydney, NSW 2109, AustraliabDepartment of Veterinary Anatomy and Pathology, University of Sydney, Sydney, NSW 2006, Australia

Received 20 October 1999; received in revised form 16 May 2000; accepted 11 July 2000

Abstract

DNA-mediated immunization was assessed in a murine model of equine herpesvirus 1 (EHV-1)

abortion. Whilst there are differences between the model and natural infection in the horse,

literature suggests that EHV-1 infection of pregnant mice can be used to assess the potential ability

of vaccine candidates to protect against abortion. Female BALB/c mice were inoculated twice, 4

weeks apart, with an expression vector encoding EHV-1 glycoprotein D (gD DNA). They were

mated 15 days after the second inoculation, challenged at day 15 of pregnancy and killed 3 days

later. The gD DNA-inoculated mice had fewer foetuses which were damaged or had died in utero

(6% in gD DNA, 21% vector DNA and 28% in nil inoculated groups challenged with EHV-1), a

reduction in the stunting effect of EHV-1 infection on foetuses (gD DNA: 0:40g� 0:06, vector

DNA: 0:34g� 0:10), reduced placental and herpesvirus-speci®c lung histopathology and a lower

titre of virus (TCID50�SEM/lung) in maternal lung than control groups (gD DNA 4:7� 0:3, vector

5:3� 0:2, nil 5:6� 0:2). Maternal antibody to EHV-1 gD was demonstrated in pups born to a dam

inoculated 123 days earlier with gD DNA. Although protection from abortion was incomplete,

immunization of mice with gD DNA demonstrated encouragingly the potential of this vaccine

strategy. # 2000 Elsevier Science B.V. All rights reserved.

Keywords: Equine herpesvirus 1; DNA immunization; Glycoprotein D; Murine abortion model

Veterinary Microbiology 76 (2000) 211±220

* Corresponding author. Tel.: �61-2-9850-8200; fax: �61-2-9850-8245.

E-mail address: [email protected] (J. Millar Whalley).

0378-1135/00/$ ± see front matter # 2000 Elsevier Science B.V. All rights reserved.

PII: S 0 3 7 8 - 1 1 3 5 ( 0 0 ) 0 0 2 6 2 - 5

1. Introduction

Equine herpesvirus 1 (EHV-1) is endemic worldwide, causing respiratory tract

infection, sporadic and epidemic abortion and neonatal mortality in horses (Allen and

Bryans, 1986). Vaccines are available, both attenuated and killed, but they have provided

incomplete protection against abortion (Burki et al., 1990; Gilkerson et al., 1997; Flore

et al., 1999). EHV-1 infection also produces respiratory disease and abortion in the mouse

(Awan et al., 1990, 1991; Walker et al., 1998a,b), creating models with which to evaluate

experimental vaccines. Whilst there have been many studies investigating vaccine

candidates in the respiratory model (Tewari et al., 1994; Osterrieder et al., 1995;

Neubauer et al., 1997; Stokes et al., 1997; Packiarajah et al., 1998; Zhang et al., 1998;

Walker et al., 1999), there has been only one report which examined the protective

ef®cacy of a baculovirus-expressed glycoprotein B of EHV-1 using the EHV-1-induced

abortion model (Kukreja et al., 1998).

We have shown previously that mice inoculated with DNA encoding EHV-1

glycoprotein D (gD) either alone (Ruitenberg et al., 1998, 1999) or followed by a boost

with baculovirus-expressed glycoprotein D (Ruitenberg et al., 2000) were able to clear

the virus signi®cantly faster and had reduced lung pathology compared with control mice

in the respiratory model of EHV-1 infection. This study reports on protection engendered

in mice inoculated with EHV-1 gD DNA against challenge infection during late

pregnancy in a modi®ed mouse model of EHV-1 abortion (Walker et al., 1998b).

2. Materials and methods

2.1. Mice, inoculation schedule and analysis of serum antibody before challenge

Female BALB/c mice (ARC, Canning Vale, WA) were anaesthetised and inoculated

intramuscularly (im) twice, 4 weeks apart (day 0 and day 28), with either 50 mg pRc/

CMV-gD (gD DNA) (Wellington et al., 1996) or pRc/CMV (vector DNA), as described

(Ruitenberg et al., 1998). Mice were tailbled on day 21 and day 38. Serum samples

collected from each mouse were pooled within their respective groups and the serum

antibody responses of the groups assessed by Western blotting, virus neutralising

antibody assays (Ruitenberg et al., 1998) and an EHV-1 ELISA (Ruitenberg et al., 1999).

ELISA antibody titres were expressed as mean absorbance at 490 nm of triplicate

samples used at a dilution of 1:500. A third group of mice received no inoculations and

had no blood taken.

2.2. Mating protocol and challenge

Mating of all mice began 43 days after the ®rst DNA inoculations were conducted. On

day 15 of pregnancy (Walker et al., 1998b), individual mice were anaesthetised and

challenged by intranasal inoculation with either 1�107 pfu of EHV-1 strain HVS25A or

an equivalent volume of rabbit kidney (RK) cell lysate (Walker et al., 1998a). One group

of mice was left uninoculated and unchallenged and served as a control. There were ®ve

212 C. Walker et al. / Veterinary Microbiology 76 (2000) 211±220

groups of pregnant mice Ð group 1: inoculated with gD DNA and challenged with EHV-

1 (gD/EHV), group 2: inoculated with vector DNA and challenged with EHV-1 (vector/

EHV), group 3: inoculated with gD DNA and challenged with RK cell lysate (gD/RK),

group 4: not inoculated and challenged with EHV-1 (NIL/EHV) and group 5: not

inoculated and not challenged (NIL/NIL).

2.3. Collection and analysis of data and samples after challenge

Rectal temperatures and body weights were recorded twice daily and clinical signs

of EHV-1 infection (Walker et al., 1998a) were scored (0:5 � slight; 1 � moderate;

2 � marked; 3 � severe). The total scores for individual mice were averaged for each

clinical examination and the mean of the totals used to de®ne the `mean clinical score

index' for each group. Parturition in EHV-1-infected mice may occur unpredictably and

as the normal maternal response is to eat the placentae and dead fetuses, tissue samples

are often lost. Consequently, as described in the modi®ed mouse model of EHV-1

abortion (Walker et al., 1998b), at day 18 of gestation, mice were killed and serum and

lung tissue collected as described (Walker et al., 1998a). Serum was used to determine the

virus neutralizing antibody titre for each mouse (Ruitenberg et al., 1998). The uterus was

exteriorised, the number of foetuses counted and their condition noted. Placentae were

examined for friability, haemorrhage and necrosis. Foetuses were examined for

subcutaneous haemorrhages or palour (indicative of foetal death in utero). Each placenta

and foetus was separated, weighed (if it had not died in utero) and collected into either

Bouins ®xative for histology or Dulbecco's modi®ed Eagle's medium (DMEM; Gibco

BRL, Mulgrave, Vic.) supplemented with foetal bovine serum 2% (FBS; CSL, Parkville,

Vic.) (DMEM-2) for virus isolation. The uterus was collected for histology. Foetal and

placental weights were analysed by comparing all group data pooled (Walker et al.,

1998b).

Where available, three placentae from each litter were examined histologically.

Congestion and necrosis of the middle layer of the trophoblast and necrosis of the chorion

were each graded according to the degree of abnormality as 0.5 (slight), 1 (5±25% of

tissue affected), 2 (25±50%) or 3 (>50%) and the mean scores compared amongst groups

(Walker et al., 1998b). Sections of maternal lung were scored (1 � mild; 2 � moderate

and 3 � severe) for bronchiolar epithelial necrosis and intranuclear inclusion bodies,

in¯ammatory in®ltrate and focal necrosis of the alveolar parenchyma, in¯ammatory

exudate in the airways and peribronchiolar and perivascular aggregations of mononuclear

cells. Virus titre in the lungs of the dams was calculated as TCID50/lung (Ruitenberg et al.,

1998) and compared among groups.

For virus isolation from foetal samples, methods were as described previously (Walker

et al., 1998b) except that tissues were chopped ®nely with a scalpel blade before

inoculation onto RK cells. Cells were checked daily for cytopathic effects. After 3 days,

the medium was aspirated and RK cells scraped and resuspended in H2O, heated to 958Cfor 10 min and clari®ed by centrifugation for PCR. PCR was carried out on 8 ml samples

with primers which ampli®ed the glycoprotein C region of EHV-1 essentially as described

(Lawrence et al., 1994). EHV-1 viral DNA and EHV-1 propagated in RK cells were used

as positive controls.

C. Walker et al. / Veterinary Microbiology 76 (2000) 211±220 213

2.4. Assessment of longevity of antibody response and maternal antibody transfer

Some of the immunised mice were excluded from the study because they failed to

maintain a pregnancy during the study period. On day 123 post-inoculation, which was

after the collection period had ®nished, one of these gD DNA-inoculated mice gave birth

to four pups. Serum antibody in the dam and her pups was assessed by Western blotting

and ELISA at 21 days (from four pups), 52 days (two pups) and 83 days after birth (two

pups).

3. Results

3.1. Antibody responses of mice used for challenge

On day 38, i.e. 10 days after the second inoculation with DNA, pooled serum samples

from gD DNA mice showed gD EHV-1-speci®c bands in Western blots and the virus

neutralizing antibody titre was 32, while the titre in serum of the vector DNA-inoculated

mouse group was <4. Mean anti-EHV-1 ELISA absorbance values at day 38 were 0.307

for the gD DNA-inoculated group and 0.03 for the vector DNA-inoculated group.

3.2. Challenge infection

The time between the second inoculation of DNA (day 28) and challenge with EHV-1

(or mock challenge with RK cell lysate) was variable and depended on the maintenance

of a successful pregnancy (Table 1). All mice challenged with EHV-1 developed clinical

signs of infection and the mean clinical scores index and body weight loss from each

group were similar (data not shown). A characteristic drop in rectal temperature 24 h

post-infection (pi) (Walker et al., 1998a) was observed in all groups challenged with

EHV-1 and continued until the mice were killed at day 3 pi. However, the mean rectal

temperature of the gD/EHV group (37.38C�1.1) was signi®cantly higher (P � 0:019) at

48 h pi than that of the vector/EHV group (35.58C�1.5).

The mean lung titre in the dams of the gD/EHV group was signi®cantly lower

(P � 0:038) than that of the other groups challenged with EHV-1 (Table 1). The gD/EHV

mice had less bronchiolar epithelial and alveolar necrosis and fewer bronchiolar epithelial

intranuclear inclusion bodies than the vector/EHV and NIL/EHV groups (Table 2).

Conversely, the in¯ammatory response was greater in the gD/EHV group, as were the

peribronchiolar and perivascular aggregations of mononuclear cells compared with the

vector/EHV and NIL/EHV groups.

One of ®ve gD/EHV and one of six vector/EHV mice aborted between days of 2.5 and

3 pi. Of the pregnancies which continued to examination at day 18 of gestation, death or

damage was evident in 6% (1/17) of foetuses from the gD/EHV group, in 21% (8/38) of

foetuses from the vector/EHV and in 28% (7/25) of foetuses from the NIL/EHV groups

(Table 1). Thus, gD inoculation provided some protection from the foetal damage or

death upon challenge with EHV-1 while vector DNA inoculation provided no protection

from challenge with EHV-1. There were no deaths or foetal abnormalities in either of the


groups not challenged with EHV-1 (the gD/RK or NIL/NIL groups of mice). Regardless

of immunization protocol, the mean weights of foetuses from the groups of mice

challenged with EHV-1 (gD/EHV: 0.40g�0.06; vector/EHV: 0.34g�0.10; NIL/EHV:

0.37g�0.07) were lower than those challenged with RK cell lysate or left unchallenged

Table 1

Effect of inoculation of mice with EHV-1 gD DNA following challenge with either EHV-1 or RK cell lysate at

day 15 of pregnancya

Group

Inoculation/

challenge

Mouse

number

Days between

second inoculationb

and challenge

Neutralizing

antibody

titre

Number of dead or

damaged foetuses/

number of foetuses

Log10

TCID50/lung

of dams

gD/EHV 1 39 16 0/4 4.5

2 41 0 0/6 5.3

3 71 0 14 3.7

4 40 11 Not pregnant 4.7

5 36 8 Aborted at day 18c 4.5

6 37 6 0/3 5.5

44d 6:8� 2:6e 4:7� 0:3f

vector/EHV 1 29 0 0/1 4.5

2 29 0 3/9 5.7

3 41 0 4/12 5.5

4 34 0 Aborted at day 18c 5.5

5 38 0 1/9 5.5

6 31 0 0/7 5.2

34 0 5:3� 0:2

gD/RK 1 34 4 0/3 NDg

2 33 0 0/5 ND

3 33 9.5 0/2 ND

4 32 9.5 0/8 ND

33 5:8� 2:3

NIL/EHV 1 NAh 0 2/7 5.7

2 NA 5 1/3 5.0

3 NA 0 2/3 6.5

4 NA 0 1/5 5.5

5 NA 0 1/7 5.5

1:0� 1:0 5:6� 0:2

NIL/NIL 1 NA 0 0/4 ND

2 NA 0 0/5 ND

3 NA 0 0/5 ND

4 NA 0 0/5 ND

5 NA 0 0/2 ND

a Parameters are measured from samples collected on day 18 of pregnancy.b 28 days after first inoculation with DNA.c Litter had been aborted (and consequently foetuses eaten) before euthanasia of dam.d Mean number of days between second inoculation and challenge for the group.e Mean virus neutralising antibody titres (mean�SEM) day 3 post-infection (pi).f Mean log10 TCID50/lung of infectious EHV-1 (mean�SEM) day 3 pi.g Not detectable.h Not applicable.


Table 2

Mean histological scores in the lungs from groups of BALB/c mice killed on day 18 of pregnancy

Group Bronchiolar

epithelial

necrosis

Intranuclear

inclusion

bodies

Focal

alveolar

necrosis

Total score of

herpesvirus-

specific

lesionsa

Cellular

infiltrate

in lung

parenchyma

Inflammatory

exudate in

airways

Peribronchiolar and

perivascular

mononuclear cell

aggregates

gD/EHV 1.1 0.8 0 1.9 1.7 1.9 1.8

vector/EHV 1.9 1.6 1.0 4.5 1.1 0.7 0.5

gD/RK 0 0 0 0 0.3 0 0.1

NIL/EHV 1.9 1.7 0.1 3.7 0.6 0.5 0.4

NIL/NIL 0 0 0 0 0 0 0.2

a Herpesvirus-specific lesions include bronchiolar epithelial necrosis, intranuclear inclusion bodies and focal alveolar necrosis.

21

6C

.W

alker

eta

l./Veterin

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76

(2000)

211±220

(gD/RK: 0.59g�0.09; NIL/NIL: 0.59g�0.12). However, the mean weight of foetuses

from the gD/EHV group was higher than that from the vector/EHV group. The mean total

histological scores of the placentae in the gD/EHV group (1.1) were approximately 60%

lower than the vector/EHV (2.8) and NIL/EHV groups (2.7) (Fig. 1) and the degree of

placental pathology in all parameters measured was less in mice inoculated with gD DNA

than in mice in the other groups challenged with EHV-1. Overall, litter survival (i.e.

normal foetuses at time of euthanasia) correlated strongly (r � ÿ0:994) with the absence

of placental histopathology. No histological abnormalities were detected in any uterus.

No virus was isolated from any foetus or placental sample from a dam infected with

EHV-1. The sensitivity of virus isolation was 5 pfu EHV-1. PCR did not detect EHV-1

DNA in any RK cell sample inoculated with foetus or placenta from any group. The limit

of sensitivity of the gC PCR test was 10 pfu EHV-1.

3.3. Antibody in pups from gD DNA-inoculated dam

At 3 weeks of age, serum from each of the four post-trial pups demonstrated gD-

speci®c bands on Western blots: the bands were as intense as those in a contemporaneous

sample from the dam. ELISA absorbance values of serum from two of the pups at 21 days

of age were 0.13 and 0.10, respectively, although by 52 days of age, these serum ELISA

antibody titres had dropped to 0.02 and ÿ0.01, respectively. There was a faint gD-speci®c

band on the Western blot in serum taken from both of these pups at 83 days. The ELISA

antibody titres from the dam at each of these sampling times was 0.27 at 144 days after

initial inoculation with gD DNA, 0.34 at 175 days and 0.34 at 206 days.

Fig. 1. Mean histological scores in the placentae of mice. gD/EHV: n � 13; vector/EHV: n � 13; gD/RK:

n � 10; NIL/EHV: n � 15; NIL/NIL: n � 14.


4. Discussion

This study has demonstrated that inoculation with DNA encoding EHV-1 gD

engendered an immune response in mice which, although incomplete, provided some

protection against the adverse effects on foetuses and foetal membranes of EHV-1

infection in a mouse model of EHV-1 abortion (Walker et al., 1998b). Importantly, only

6% of foetuses died or were damaged in mice inoculated with gD DNA compared with

21% of those inoculated with vector DNA and 28% of those not given DNA before

challenge. The mean weight of foetuses in the gD DNA group was higher (0.40g�0.06)

than that of the vector DNA group (0.34g�0.10) although mean weights of groups of

foetuses from EHV-1 challenged mice were lower than those not challenged with EHV-1.

This suggested that inoculation with gD DNA afforded some protection of mouse

foetuses against the adverse effects of EHV-1 infection seen in the dams in this model. As

well, a correlation between foetal protection and reduced placental pathology was

observed in the gD DNA-inoculated group. As seen in this study where no EHV-1 was

isolated from any foetus or placenta from an EHV-1 infected dam, vascular abnormalities

in the murine placenta in the absence of viral infection of the foetus and/or placenta are

not uncommon (Walker et al., 1998b), suggesting that EHV-1-induced abortion in the

mouse sometimes may be due to maternal factors alone. This hypothesis is supported by a

study which showed that, following experimental EHV-1 infection in pregnant mares late

in gestation, 44% of aborted equine foetuses were virologically negative and showed no

characteristic herpesvirus-speci®c histopathological lesions (Smith et al., 1992). The

®ndings here of adverse effects of the large challenge dose of EHV-1 and its effects on the

rectal temperature of the dams and on the foetal weights also supported this hypothesis.

While all challenged mice became ill as a consequence of virus challenge (shown by a

drop in body temperature and high clinical score indices) and foetal weights of all

challenged mice were less than those in the control groups, these effects were less in the

gD DNA inoculated group suggesting that inoculation with gD DNA afforded some

protection of the foetus from these adverse maternal factors.

In an earlier study, using the murine respiratory model of EHV-1 infection, where mice

were inoculated either once or twice with gD DNA, the antibody response increased until

the time of last sampling (8 and 23 weeks, respectively) (Ruitenberg et al., 1998). In the

present study, where one mouse was followed for an extended period, the antibody level

remained constant between days 144 and 206 after gD DNA was ®rst inoculated.

Together, these ®ndings suggest that the humoral response engendered by intramuscular

inoculation with gD DNA may be long-lived, possibly because antigenic stimulation

continues throughout the life of myocytes expressing the antigen on their surface

(Donnelly et al., 1997). Although in the current study cell-mediated responses were not

measured due partly to the complexity of the experimental design, previous results have

shown that mice inoculated with the EHV-1 gD DNA induced a lymphoproliferative

response and engendered a Th1 helper lymphocyte pro®le. Our study has also shown that

anti-gD antibody was transferred to pups via colostrum. Similarly, pups of mice

immunised with DNA encoding pseudorabies virus immediate early protein acquired

maternal antibody by passive transfer and 28% of such pigs survived a lethal challenge at

14 weeks of age (Chang et al., 1998).


While the results presented here show that inoculation with gD DNA afforded some

protection against loss of foetal viability/abnormality, the protection was not absolute.

This may be explained in part at least by the dose of EHV-1 needed to induce abortion in

this model (1�107 pfu HVS25A) compared with the EHV-1 respiratory disease model

(5�105 pfu). Importantly, all mice showed severe clinical signs, had virus histopathology

and cleared virus more slowly than those challenged in the respiratory model of EHV-1

infection. This high challenge dose may have masked some of the protective ef®cacy of

vaccination and may diminish the apparent usefulness of this approach to assess potential

vaccines for the horse.

In order to prevent EHV-1 abortion, a vaccine must prevent severe disease and/or

viraemia and the associated vascular effects on the placenta, endometrium and

transplacental transfer of virus to the foetus. Therefore, it must provide a response

which restricts viral replication to respiratory surfaces. This work, combined with the

earlier ®ndings of viral clearance associated with gD DNA in the respiratory model,

suggests that this approach has merit as a potential vaccine candidate or vaccine

component.

Acknowledgements

This project was supported in part by the Australian Research Council and the Rural

Industries Research and Development Corporation with collaborative support from CSL

Ltd., and by a Macquarie University Research Grant. K.M. Ruitenberg was the recipient

of an Australian Postgraduate Award. The authors thank Norbert Lambert and Herbert

Ayala for their care of mice, and Karen Wadwell and Elaine Chew for preparation of

histological sections.

References

Allen, G.P., Bryans, J.T., 1986. Molecular epizootiology, pathogenesis, and prophylaxis of equine herpesvirus-1

infections. Prog. Vet. Microbiol. Immunol. 2, 78±144.

Awan, A.R., Chong, Y.C., Field, H.J., 1990. The pathogenesis of equine herpesvirus type 1 in the mouse: a new

model for studying host responses to the infection. J. Gen. Virol. 71, 1131±1140.

Awan, A.R., Gibson, J.S., Field, H.J., 1991. A murine model for studying EHV-1-induced abortion. Res. Vet. Sci.

51, 94±99.

Burki, F., Rossmanith, W., Nowotny, N., Pallan, C., Mostl, K., Lussy, H., 1990. Viraemia and abortions are not

prevented by two commercial equine herpesvirus-1 vaccines after experimental challenge of horses. Vet.

Quart. 12, 80±86.

Chang, S.W., Bu, J., Rompato, G., Garmendia, A.E., 1998. A vector DNA vaccine encoding pseudorabies virus

immediate early protein demonstrates partial protection in mice against lethal virus challenge. Viral

Immunol. 11, 27±36.

Donnelly, J.J., Ulmer, J.B., Shiver, J.W., Liu, M.A., 1997. DNA vaccines (review). Annu. Rev. Immunol. 15,

617±648.

Flore, P.H., Minke, J.M., Mumford, J.A., Hannant, D., Jessett, D., Vandehoek, J., Westrate, M.W., 1999. Studies

on the ef®cacy of an inactivated, carbomer-adjuvanted equine herpesvirus vaccine in pregnant mares in the

face of a challenge with an abortigenic strain of EHV-1. In: Proceedings of the Eight International

Conference on Equine Infectious Diseases, Vol. VIII, pp. 422±423.


Gilkerson, J.R., Love, D.N., Whalley, J.M., 1997. A review of equine herpesvirus vaccine: from the traditional to

the experimental. Aust. Equine Vet. 15, 27±32.

Kukreja, A., Walker, C., Fitzmaurice, T., Awan, A., Love, D.N., Whalley, J.M., Field, H.J., 1998. Protective

effects of equine herpesvirus 1 (EHV-1) glycoprotein B in a murine model of EHV-1-induced abortion. Vet.

Microbiol. 62, 303±311.

Lawrence, G.L., Gilkerson, J., Love, D.N., Sabine, M., Whalley, J.M., 1994. Rapid, single-step differentiation of

equid herpesviruses 1 and 4 from clinical material using the polymerase chain reaction and virus-speci®c

primers. J. Virol. Meth. 47, 59±72.

Neubauer, A., Beer, M., Brandmuller, C., Kaaden, O.R., Osterrieder, N., 1997. Equine herpesvirus 1 mutants

devoid of glycoprotein B or M are apathogenic for mice but induce protection against challenge infection.

Virology 239, 36±45.

Osterrieder, N., Wagner, R., Brandmuller, C., Schmidt, P., Wolf, H., Kaaden, O.R., 1995. Protection against

EHV-1 challenge infection in the murine model after vaccination with various formulations of recombinant

glycoprotein gp14 (gB). Virology 208, 500±510.

Packiarajah, P., Walker, C., Gilkerson, J.R., Whalley, J.M., Love, D.N., 1998. Immune responses and protective

ef®cacy of recombinant baculovirus expressed glycoproteins of equine herpesvirus 1 (EHV-1) gB, gC and

gD alone or in combinations in BALB/c mice. Vet. Microbiol. 61, 261±278.

Ruitenberg, K., Walker, C., Wellington, J., Love, D.N., Whalley, J.M., 1998. DNA-mediated immunization with

glycoprotein D of equine herpesvirus 1 (EHV-1) in a murine model of EHV-1 respiratory infection. Vaccine

17, 237±244.

Ruitenberg, K.M., Walker, C., Wellington, J.E., Love, D.N., Whalley, J.M., 1999. Potential of DNA-mediated

vaccination for equine herpesvirus 1. Vet. Microbiol. 68, 35±48.

Ruitenberg, K.M., Walker, C., Wellington, J.E., Love, D.N., Whalley, J.M., 2000. A prime-boost immunization

strategy with DNA and recombinant baculvirus-expressed protein enhances protective immunogenicity of

glycoprotein D of equine herpesvirus 1 in naõÈve and infection-primed mice. Vaccine 18, 1367±1373.

Smith, K.C., Whitwell, K.E., Binns, M.M., Dolby, C.A., Hannant, D., Mumford, J.A., 1992. Abortion of

virologically negative foetuses following experimental challenge of pregnant pony mares with equid

herpesvirus 1. Equine Vet. J. 24, 256±259.

Stokes, A., Cameron, R.S., Marshall, R.N., Killington, R.A., 1997. High level expression of equine herpesvirus 1

glycoproteins D and H and their role in protection against virus challenge in the C3H (H-2Kk) murine model.

Virus Res. 50, 159±173.

Tewari, D., Whalley, J.M., Love, D.N., Field, H.J., 1994. Characterization of immune responses to baculovirus-

expressed equine herpesvirus type 1 glycoproteins D and H in a murine model. J. Gen. Virol. 75, 1735±1741.

Walker, C., Packiarajah, P., Gilkerson, J.R., Love, D.N., Whalley, J.M., 1998a. Primary and secondary infection

of mice with equine herpesvirus 1 strain HVS25A. Virus Res. 57, 151±162.

Walker, C., Perotti, V.M., Love, D.N., Whalley, J.M., 1998b. Infection with equine herpesvirus 1 (EHV-1) strain

HVS25A in pregnant mice. J. Comp. Path. 120, 15±27.

Walker, C., Love, D., Whalley, J.M., 1999. Comparison of the pathogenesis of acute equine herpesvirus 1

(EHV-1) infection in the horse and the mouse model: a review. Vet. Microbiol. 68, 3±13.

Wellington, J.E., Lawrence, G.L., Love, D.N., Whalley, J.M., 1996. Expression and characterization of equine

herpesvirus 1 glycoprotein D in mammalian cell lines. Arch. Virol. 141, 1785±1793.

Zhang, Y.F., Smith, P.M., Tarbet, E.B., Osterrieder, N., Jennings, S.R., O'Callaghan, D.J., 1998. Protective

immunity against equine herpesvirus type 1 (EHV-1) infection in mice induced by recombinant EHV-1 gD.

Virus Res. 56, 11±24.


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