Evaluation of a recombinant vaccinia virus containing pseudorabies (PR) virus glycoprotein genes gp50, gII, and gIII as a PR vaccine for pigs.
ABSTRACT Pigs vaccinated twice intramuscularly with a highly attenuated strain of vaccinia virus (NYVAC) containing gene inserts for pseudorabies virus (PRV) glycoproteins gp50, gII, and gIII produced neutralizing antibodies for PRV and were less clinically affected than were nonvaccinated pigs following oronasal exposure to virulent PRV. Also, following oronasal exposure to virulent PRV the duration of virulent virus shedding by pigs that had been vaccinated intramuscularly with the recombinant virus was statistically less (p < 0.05) than that of nonvaccinated pigs and like that of pigs vaccinated twice intramuscularly with inactivated PR vaccine. Intramuscular vaccination with the recombinant virus was compatible with the most commonly used differential diagnostic tests, namely those based on PRV glycoproteins gX and gI. Serum antibodies for these glycoproteins were absent from the sera of all pigs before and after vaccination with recombinant virus; whereas, they were present in the sera of all of the same pigs after they were exposed to virulent PRV. In contrast to the effectiveness of the recombinant virus administered intramuscularly, neither serum antibody nor clinical protection against PRV was detected when aliquots of the same recombinant virus preparation were administered either orally or intranasally. The latter finding suggests that recombinant virus replicates poorly, if at all, at these sites. If so, the dissemination of recombinant virus from vaccinated pigs to nonvaccinated pigs or other animals in contact seems unlikely.
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ABSTRACT: The glycoprotein gB of pseudorabies virus (PrV) was expressed in various mammalian cells by a recombinant baculovirus carrying the PrV gB gene under the control of the CAG promoter. When the recombinant baculovirus was inoculated into the stable porcine kidney cell line CPK, expression of PrV gB was detected by immunofluorescent antibody analysis and a 155 kDa of protein, which has the same molecular mass as the native PrV gB, was detected by Western blotting. High levels of expression of PrV gB were observed in BHK-21, HmLu-1 and SK-H cell lines. Furthermore, anti-PrV gB-specific antibodies against PrV gB protein were detected by the enzyme-linked immunosorbent assay in mice inoculated the recombinant baculovirus. The recombinant baculovirus containing the PrV glycoprotein gB gene under the CAG promoter could be a candidate for a pseudorabies vaccine.Veterinary Microbiology 08/1999; 68:197-207. · 2.73 Impact Factor
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ABSTRACT: The present study demonstrates the protective potential of novel baculovirus recombinants, which express the glycoproteins gB, gC, or gD of Pseudorabies virus (PRV; Alphaherpesvirus of swine) and additionally contain the glycoprotein G of Vesicular Stomatitis Virus (VSV-G) in the virion (Bac-G-PRV). To evaluate the protective capacity, mixtures of equal amounts of the PRV gB-, gC-, and gD-expressing baculoviruses were used for immunization. Three intramuscular immunizations with that Bac-G-PRV mixture could protect mice against a lethal PRV challenge infection. To achieve complete protection high titers of Bac-G-PRV and three immunizations were necessary. This immunization with Bac-G-PRV resulted in the induction of high titers of PRV-specific serum antibodies of the IgG2a subclass and of interferon (IFN)-gamma, indicating a Th1-type immune response. Moreover, splenocytes of immunized mice exhibited natural killer cell activity accompanied by the production of IFN-alpha and IFN-gamma. Collectively, the presented data demonstrate for the first time that co-expression of VSV-G in baculovirus recombinant vaccines can improve the induction of a protective immune response against foreign antigens.Vaccine 07/2009; 27(27):3584-91. · 3.49 Impact Factor
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ABSTRACT: Adenovirus and poxvirus recombinant vectors are more and more used as live experimental vaccines. In order to compare the efficacy of these vectors to elicit serological response and protection against challenge, two recombinants carrying the same gene (pseudorabies virus gD) were used as experimental vaccines in mice, a permissive species to pseudorabies infection. Two routes were tested: intramuscular (i.m.) and intranasal (i.n.) in order to try to stimulate general and mucosal immune responses. Several doses ranging from 10(2.9) to 10(8.9) TCID50, depending on the vaccines were tested. The estimated log 10 (PD50) for the i.m. route were 7.1 +/- 0.2 for the adenovirus vector (Ad-gD), and 7.6 +/- 0.2 for the Nyvac vector (vP900). For the i.n. route, log 10 (PD50) of Ad-gD was 7.1 +/- 0.2, and was higher than 7.9 for vP900. While the adenovirus vector proved more efficient than the poxviral vector to elicit antibody response, only a slight difference was observed when comparing the survival times of animals after challenge. Adenovirus was found better only for the 10(7.9) TCID50 dose, when inoculated i.m. Intranasal vaccination appeared efficient only with the adenovirus vector for the TCID50 10(8.9) dose.Vaccine 09/1996; 14(11):1083-7. · 3.49 Impact Factor
Arch Virol (1994) 134:259-269
© Springer-Verlag t994
Printed in Austria
Evaluation of a recombinant vaccinia virus containing pseudorabies (PR)
virus glycoprotein genes gp50, glI, and glII as a PR vaccine for pigs
w. L. Mengeling, Susan L. Brockmeier, and K. M. Lager
Virology Swine Research Unit, National Animal Disease Center, USDA, Agricultural
Research Service, Ames, Iowa, U.S.A.
Accepted September 29, 1993
Summary. Pigs vaccinated twice intramuscularly with a highly attenuated
strain of vaccinia virus (NYVAC) containing gene inserts for pseudorabies virus
(PRV) glycoproteins gpS0, gII, and gIII produced neutralizing antibodies for
PRV and were less clinically affected than were nonvaccinated pigs following
oronasal exposure to virulent PRV. Also, following oronasal exposure to
virulent PRV the duration of virulent virus shedding by pigs that had been
vaccinated intramuscularly with the recombinant virus was statistically less
(p < 0.05) than that of nonvaccinated pigs and like that of pigs vaccinated twice
intramuscularly with inactivated PR vaccine. Intramuscular vaccination with
the recombinant virus was compatible with the most commonly used differential
diagnostic tests, namely those based on PRV glycoproteins gX and gI. Serum
antibodies for these glycoproteins were absent from the sera of all pigs before
and after vaccination with recombinant virus; whereas, they were present in
the sera of all of the same pigs after they were exposed to virulent PRV. In
contrast to the effectiveness of the recombinant virus administered intra-
muscularly, neither serum antibody nor clinical protection against PRV was
detected when aliquots of the same recombinant virus preparation were admin-
istered either orally or intranasally. The latter finding suggests that recombinant
virus replicates poorly, if at all, at these sites. If so, the dissemination of
recombinant virus from vaccinated pigs to nonvaccinated pigs or other animals
in contact seems unlikely.
Pseudorabies (PR) is a contagious and sometimes fatal disease caused by a
herpesvirus, pseudorabies virus (PRV), of the subfamily Alphaherpesvirinae .
It affects several species of wild and domesticated animals but is most common
in pigs, which also serve as the major interepizootic reservoir and the primary
means for dissemination of the causative virus [2-4].
260 W.L. Mengeling et al.
Presently the control of PR of pigs is based largely on the use of vaccines
that contain either the entire complement of virus-coded proteins (inactivated
and attenuated virus vaccines) [2, 5] or selected viral glycoproteins (subunit
vaccine) . All are effective in reducing the clinical effects of the disease.
Moreover, attenuated and inactivated vaccines prepared from deletion-mutant
strains of PRV, as well as subunit vaccines, can be used in conjunction with
appropriate diagnostic tests to identify pigs exposed to virulent, wild-type PRV.
Differentiation depends on the detection of antibodies for one or more viral
proteins associated only with virulent virus. The most commonly used tests
are based on glycoprotein I (gI)  or glycoprotein X (gX)  which are, respec-
tively, structural and nonstructural glycoproteins of PRV . The primary
indication for differential testing is when vaccines are used in conjunction with
PR eradication programs (such as those now in progress in the United States
and Europe) because, regardless of vaccination history, all pigs that survive
exposure to virulent PRV are likely to become latently-infected carriers with
the potential for virus reactivation and shedding .
Recently another type of vaccine was developd by using a highly attenuated
strain (NYVAC) of vaccinia virus as a vector for selected genes of PRV .
Three constructs of this vector have already been tested for their immunogeni-
city in pigs . They differed in that each contained a single gene for one of
the three glycoproteins of PRV that are believed to be important immunogens
involved in clinical protection, namely, gp50, gII, and gIII. Although the
gp50-vectored vaccine appeared to be the most effective, all three stimulated
a humoral, virus-neutralizing (VN) antibody response and some degree of
In the study reported here we vaccinated pigs with the NYVAC vector
containing PRV genes for gp50, gII, and gIII to test the combined effect of
these antigens on immunity. We also compared the effects of administering the
recombinant vaccine intramuscularly (IM), intranasally, or orally, and investi-
gated the compatibility of NYVAC-vectored vaccination with two of the
differential diagnostic tests (gI and gX) most often used in conjunction with
the PR eradication program in the United States.
Materials and methods
Thirty-two 5-week-old pigs were purchased from a commercial herd that was free of
infection with PRV. On the day they were delivered to our facility they were weighed, ear
tagged, and allocated to five treatment groups (eight pigs for group I and six pigs/group
for groups 2 through 5), so that each group was comprised of pigs of about the same
weight distribution and, thus, the same average weight. Pigs of group 1 were kept as
nonvaccinated controls, whereas all pigs of groups 2 through 5 were vaccinated twice at
a 28-day interval. Pigs of group 2 were vaccinated (2 ml/dose) IM with inactivated PRV.
Pigs of groups 3 through 5 were vaccinated (2 ml/dose) IM (group 3), intranasally (group 4),
or orally (group 5) with the NYVAC strain of vaccinia vector containing genes for PRV
glycoproteins gp50, gII, and gIII. Infectivity titers and additional details relative to these
Vaccinia-virus-vectored vaccine for pseudorabies 261
vaccines are presented in a subsequent section on "Vaccine preparation." The treatment
schedule was: day- 14 to day 0, all pigs were acclimated to our isolation facilities; day 0,
first vaccination; day 28, second vaccination; day 56, challenge-exposure, i.e., challenge of
immunity by oronasal exposure of each pig to 2 ml of virulent PRV containing 2.8 x 108
plaque forming units (PFU) of virus per ml; day 84, euthanasia by an overdose of barbiturate
and necropsy. A blood sample was collected from each pig on days 0, 28, 56, and 84, and
the corresponding sera were titered for VN antibodies for PRV. Sera collected on days 0
and 56 were also titered for VN antibodies for vaccinia virus (both the parent NYVAC
strain and the recombinant virus). Selected sera also were tested for antibodies for gI and
gX. All pigs were weighed at the time they were received on day- 14 and on days 0, 3, 5,
10, 14, 56, 59, 63, 66, 70, and 74. Body (rectal) temperatures were recorded for all pigs on
days 0, 3, 5, 10, 14, 56, 57 through 67, 70, and 74. Oropharyngeal swabs were collected
from all pigs on days 56, 58, 60, 63, 66, 70, and 74. Swabs were tested for the presence and
titer of PRV shedding.
Isolation and observation of pigs
Pigs were kept in four isolation rooms throughout the study. The arrangement from the
time they were received until 1 day after they were exposed to virulent PRV (an interval
of about 10 weeks, see above) was: room 1, six pigs of group 1 and all pigs of group 2;
room 2, two pigs of group 1 and all pigs of group 3; rooms 3 and 4, all pigs of groups 4
and 5, respectively. On the first day after exposure to virulent PRV, two pigs of group t,
room t, were moved to room 3, and two pigs of group 1, room 1, were moved to room
4, so that each isolation room thereafter contained two pigs of group 1 and all pigs of one
other treatment group. All pigs were observed daily throughout the experiment.
Virus neutralizing antibody titrations
Titers of serum VN antibodies for PRV and vaccinia virus were determined by methods
previously described .
Bovine embryonic spleen (BESp) cells  were used to test oropharyngeal swabs for PRV
and a porcine kidney (PK-15) established cell line was used for all other laboratory
procedures involving PRV. Primary chicken embryo fibroblasts were used to propagate
the NYVAC strain of vaccinia virus for vaccine production and BESp cells were used for
all other laboratory procedures involving vaccinia virus. All cells were grown as stationary
monolayer cultures in medium that consisted of Eagle's minimal essential medium (EMEM)
supplemented with 0.5~ lactalbumin hydrolysate, gentamicin sulfate (50 gg/ml) and either
5~o fetal bovine serum (FBS) for PK-15 cells or 10~o FBS for all other cell types.
The attenuated, commercially available, Tolvid strain of PRV containing deletions in its
thymidine kinase (TK) and gX genes  was used to prepare inactivated PRV vaccine.
The virulent Indiana-Funkhauser strain of PRV was used to challenge immunity of pigs
and to test sera for VN activity.
262 W.L. Mengeling et al.
The NYVAC strain of vaccinia virus was derived by genetically engineering the deletion
of putative virulence and host range genes from the Copenhagen strain of vaccinia virus
. The NYVAC strain containing insertions of PRV genes for gp50, gII, and gIII was
used to prepare vaccinia-vectored PRV vaccine. The NYVAC strain, both with and without
PRV gene insertions, was used to test sera for VN activity.
Inactivated PRV vaccine used in this study was prepared and evaluated previously .
The infectivity titer prior to inactivation was 7 x 108 PFU/ml. It was used undiluted except
for the addition of 0.! volume of aluminium hydroxide as an adjuvant.
The vaccinia-vectored PRV vaccine virus was prepared by infecting stationary, monolayer
cultures of primary chicken embryo fibroblast at a multiplicity of infection of about 0.1.
Two days later, when virus-induced cytopathic effects (CPE) were extensive, the cultures
were frozen (-80°C) and thawed twice and the cell culture medium was clarified by
centrifugation (500 x g, 15 min). The clarified cell culture medium was used undiluted as
vaccine. Its infectivity titer was 10 v median cell culture infective doses (CCIDso)/ml.
Expression of all three PRV gtycoproteins by the vaccinia vector was confirmed by
propagating an aliquot of the vaccine in BESp and porcine lung celt cultures on coverslips
in Leighton tubes and testing replicate, infected cultures by indirect immunofluorescence
microscopy against primary sera specific for gII, gIII, or gp50.
Differential diagnostic tests
Selected sera were tested for antibodies for PRV glycoproteins gI and gX using test kits
licensed for use in the PR eradication program in the United States (HerdChek: Anti-PRV-gI
and HerdChek: Anti-PRV-gpX, IDEXX Laboratories Inc., One IDEXX Drive, Westbrook,
ME, U.S.A.). All sera collected from pigs on days 56 and 84 were first tested for antibodies
for gI and gX in a regulatory laboratory where such testing is routinely performed
(Diagnostic Virology Laboratory, National Veterinary Services Laboratories, USDA,
Animal and Plant Health Inspection Service, Ames, IA, U.S.A.). After obtaining the results
of these tests we retested all of the same sera plus sera collected from the same pigs on
day 0 for the presence of antibodies for gX.
Oropharyngeal swab samples
Procedures for collection of oropharyngeal swab samples, for detection and titration of
associated PRV, and for statistical analyses of virus shedding among groups were as
previously described .
After the first vaccination (days 0 through 28)
All pigs remained clinically normal during the 28 days immediately following
the first vaccination. Sera collected from pigs just before the first vaccination
(day 0) were free of VN antibody for both PRV and vaccinia virus. Those
collected from pigs of groups 1, 3, 4, and 5 at 28 days after the first vaccination
also were free of VN antibody for PRV, whereas all of those collected at the
same time from pigs of group 2 had VN antibody (Table 1).
Vaccinia-virus-vectored vaccine for pseudorabies 263
Table 1. Virus neutralizing (VN) antibody titer.s for pseudorabies virus
(PRV) in sera obtained from pigs before and after vaccination and
Group Vaccine Route of
Days after 1st vaccination"
0 28 56 84
inactivated PRV intramuscular <2
vaccinia vector c intramuscular <2
vaccinia vector c oral
vaccinia vectoff intranasal
.... <2 b <2 <2 9.75
8.5 9.7 13.3
"Swine were vaccinated on days 0 and 28, and exposed to virulent PRV
on day 56
b Geometric mean titer of VN antibodies
c Recombinant vaccinia virus containing PRV glycoprotein genes gp50,
gII, and gIII
After the second vaccination (days 28 through 56)
All pigs remained clinically normal during the 28 days immediately following
the second vaccination. Sera collected from pigs of groups 1, 4, and 5 just before
their immunity was challenged at day 56 were free of VN antibody for PRV.
Conversely, sera collected at the same time from pigs of group 3 had VN
antibody for PRV, and those collected from pigs of group 2 had an increase
in VN antibody for PRV (Table 1). Sera collected from pigs of group 3 on day 56
(i.e. 28 days after the second dose of vaccinia-vectored vaccine) appeared to have
a low level of inhibitory activity for vaccinia virus in that the vaccinia-virus-
induced CPE usually developed slower at low serum dilutions than it did when
the virus was reacted with the same dilutions of prevaccination (day 0) sera from
the same pigs. However, the inhibitory activity was never sufficient to block
infectivity in all of the cultures even at the lowest dilution (1:2) of serum tested.
There was no apparent difference between the reactivity of sera with the NYVAC
strain of vaccinia virus either with or without PRV gene insertions.
After challenge of immunity with virulent PRV (days 56 through 84)
All pigs of all treatment groups had clinical signs (inappetence, listlessness,
respiratory distress, and in some cases incoordination) following oronasal
exposure to virulent PRV. In general, the severity of signs paralleled the increase
in body temperatures (Fig. 1). Pigs of groups 2 and 3 appeared less severely
affected than did those of groups t, 4, and 5. One pig of group 4 died 7 days
after challenge-exposure, whereas all others recovered. Pigs of groups 2 and
3 gained body weight during the 7 days immediately following challenge-
exposure, whereas those of groups 1, 4, and 5 lost weight during the same
interval (Fig. 2). All groups had gained weight by the 14th day after challenge-
264 W.L. Mengeling et al.
............... Group 2
.......... Group 3
Group 5 k
X_ v \ ...........
38.5 56 517 5'8 519 610 6~1 6J2 613 6'4 6'5 6~6 617 618 6~9 710 7'1 712 713 714
Fig. 1. Mean body (rectal) temperatures of pigs after challenge of immunity by oronasal
exposure to virulent pseudorabies virus on day 56
50 1 Group 1
.................... Group 2
" . . . . . . . . .
404 s..#.... "
57 5'8 5'9 610 6'1 6~2 613 614 6'5 6'6 6'7 6'8 6'9 710 7'1 7'2 7~3 7~4
Mean percent weight gain of pigs after challenge of immunity by oronasal exposure
to virulent pseudorabies virus on day 56
Vaccinia-virus-vectored vaccine for pseudorabies
Table 2. Duration of shedding of virulent pseudorabies virus (PRV)
following challenge exposure
Group Vaccine Route of
vaccinia vector b
vaccinia vector b
vaccinia vector b
3.75 (0.31) a
a Mean (standard deviation of the mean); values based on the days
oropharyngeal swabs were collected, namely days 56, 58, 60, 63, 66, 70,
b Recombinant vaccinia virus containing PRV glycoprotein genes gp50,
gII, and glII
exposure. All pigs had serum VN antibody for PRV on the 28th day after
challenge-exposure (day 84); however, the highest titers were in sera of pigs in
which antibodies had been raised previously by vaccination (Table 1). All pigs
of all groups shed PRV after challenge exposure. For the 6 days on which
samples were collected after challenge exposure (i.e., days 58, 60, 63, 66, 70,
and 74 of the experiment and days 2, 4, 7, 10, 14, and 18 after challenge) the
mean number of days of shedding ranged from 2.3 days for pigs of group 3 to
4.2 days for pigs of group 5 (Table 2). The difference in the duration of shedding
between either group 2 or 3 and any of the other groups was statistically
significant (P < 0.05). On the other hand there was no statistical significance
between the duration of shedding of groups 2 and 3.
Differential diagnostic tests
Sera collected on day 56 (just before challenge-exposure) from all pigs of groups
1, 3, 4, and 5 were negative by both the gI and gX differential diagnostic tests.
Sera collected at the same time from pigs of group 2 were all positive by the
gI differential diagnostic test and two were positive by the gX differential
diagnostic test. All of the test results were clearly either positive or negative
except for one of the two sera that were positive for antibodies for gX. The
reading (optical density) associated with this serum was just low enough for
the serum to be classified as positive. The same results were obtained when
additional aliquots of the sera collected on day 56 were retested for antibodies
for gX, except that the optical density associated with the serum that previously
had been just low enough to be classified as positive was, on retest, just sufficient
for this serum to be classified as negative, i.e., free of antibodies for gX. Sera
collected on day 84 (28 days after challenge-exposure) all contained antibodies
for both gX and gI. The same result was obtained when all of these sera were
266 w.L. Mengeling et al.
tested a second time for antibodies for gX. In contrast, none of the sera collected
from the same pigs on day 0 (just before the first vaccination) contained
antibodies for gX.
Pigs vaccinated IM either with inactivated PRV (group 2) or with a vaccinia
virus vector containing PRV genes for gII, gIII, and gp50 (group 3) developed
immunity to subsequent challenge exposure to virulent PRV. These vaccination
procedures appeared to be equally effective in inhibiting shedding of virulent
virus after challenge exposure; however, a comparison of temperature responses
(Fig. 1) and weight gains (Fig. 2) and our assessment of clinical signs indicated
that greater clinical protection was provided by vaccination with inactivated
PRV. Whether these differences would be evident in the field where exposure
to virulent PRV is likely to be much less than it was in this study, or if they
could be affected appreciably by changes in the vaccination procedure, such
as by increasing the dose of the vaccinia-vectored vaccine, remains to be
determined. Moreover, we emphasize that the inactivated PRV vaccine to which
the recombinant was compared may be an exceptionally effective immunogen and
perhaps more effective than commercially available inactivated vaccines. During
a previous study we found that a single dose of this vaccine stimulated a higher
titer of serum VN antibody than did a similar administration of the same strain
of attenuated virus .
By comparing the results of this study with those of a previous study 
wherein we tested the same glycoproteins individually, it appears that the
combination (gII, gIII, and gp50) is more immunogenic than either gII or gIII
alone; however, there is, as yet, no clear evidence from out studies that it is
appreciably more immunogenic than gp50 alone. On the basis of serum VN
antibody titers and clinical protection against virulent PRV, pigs vaccinated
IM with the NYVAC vector containing the gene for gp50  responded at
least as well as those vaccinated with the same vector containing all three genes.
Because the vaccines were evaluated in different studies, it is possible that other
variables affected the results. Moreover, in a study by others [t5] in which the
same PRV glycoprotein genes were expressed either alone or in combination
via a less attenuated vaccinia vector, there was no evidence that gp50 was any
more immunogenic than gII or gIII for either mice or pigs that were vaccinated
and subsequently exposed to virulent PRV. In fact, mice were protected better
by a vector containing gII and gIII than by any of the vector-glycoprotein
combinations containing gp50. An explanation for these apparent discrepancies
may depend on the relative and/or absolute levels of expression of the different
glycoprotein genes by the various recombinants. Nevertheless, if our obser-
vations prevail and it is found that the immunity provided by the gp50 vector
is sufficient for field use in pigs, other options concerning differential diagnostic
tests become obvious, namely, the gp50 vaccine would probably be compatible
with differential testing based on other glycoproteins of PRV, such as gIII and
Vaccinia-virus-vectored vaccine for pseudorabies 267
gII. The latter might be particularly useful in that it is both an essential
glycoprotein and an excellent antigen which conceivably would always induce
a detectable level of homologous serum antibody regardless of the strain of
PRV to which a pig had been exposed.
In contrast to the immune response of pigs following IM administration of
the vectored vaccine, there was no evidence that either intranasal or oral
administration of the same vaccine raised any immunity to PRV (Table 1, Figs. 1
and 2). In this regard the immune response to the vectored PRV vaccine was
like that reported previously for inactivated PRV vaccine and unlike the
enhanced immunity induced by oronasal administration of live, attenuated
PRV . Assuming that the vectored vaccine is infectious for porcine cells in
vivo, as we know it to be in vitro, we are left with the possibility that susceptible
cell populations are limited or inaccessible by oronasal routes of exposure. If
so, the potential for IM-vaccinated swine to disseminate the vector through
oronasal secretions to other animals or people in contact would seem to be
In general, the results of differential testing of sera for antibody to PRV glyco-
proteins gX and gI were consistent with the histories of exposure through vacci-
nation or challenge or both. Notably, the vaccinia-vectored vaccine appeared
to be fully compatible with gX and gI differential tests currently used in the
eradication program for PR in the United States. Sera from nonvaccinated
pigs and from pigs that were vaccinated with vectored vaccine were free of anti-
body for both glycoproteins until after their immunity was challenged with
virulent (gX +, gI +) PRV. After challenge-exposure, sera from all of the same
pigs contained antibody for both glycoproteins. In contrast, all sera from pigs
vaccinated with the inactivated Tolvid (gX-, gI +) contained antibodies for
gI after vaccination, as well as after challenge exposure. Prevaccination sera of
this group were not tested for antibodies to gI; but since all of the other pigs of
the experiment were free of antibodies to gI even after vaccination, we can
logically assume that seroconversion to gI was due to vaccination. The only
unexpected findings of the study in regard to differential testing were the positive
test results of gX testing of sera of two pigs that had been vaccinated with
inactivated PRV. Because the inactivated vaccine was prepared from a gX
deletion mutant, none of the vaccinated pigs should have had post-vaccination
antibody to gX unless there is an undeleted portion of the gX gene that codes
for the epitope corresponding to the monoclonal antibody used in the test kit.
If so, it is not clear why post-vaccination serum from other pigs of group 2
did not also have a clearly positive reaction for antibodies for gX, especially
since two doses of the vaccine were administered prior to collection of sera at
day 56. Whatever, the fundamental reason(s) for these inconsistencies in gX
test results, we assume that they reflect an incompatibility between the test and
this particular attenuated vaccine. Previous recognition of the problem is
suggested by the fact that the HerdChek: Anti-PRV-gpX differential test is not
recommended by its manufacturer for use with Tolvid vaccine.
268 W.L. Mengeling et al.
The authors thank Dr. M.L. Frey and Mr. K. A. Eernisse, Diagnostic Virology
Laboratory, National Veterinary Services Laboratories, USDA, Animal and Plant Health
Inspection Service, Ames, Iowa 50010, for testing sera for antibodies to gI and gX, and A.C.
Vorwald, T. E. Rahner, D. S. Adolphson, and D. V. Hackbarth for technical assistance.
This study was done as part of a formal Cooperative Research and Development Agreement
between the ARS, USDA, and the Virogenetics Corporation, Troy, New York. The initial
stocks of the NYVAC strain of vaccinia virus, both with and without gene inserts for
pseudorabies virus glycoproteins, were provided by Virogenetics.
1. Mettenleiter TC (1991) Molecular biology of pseudorabies (Aujeszky's disease) virus.
Comp Immunol Microbiol Infect Dis 14:151-163
2. Wittmann G, Rziha HJ (1989) Aujeszky's disease (pseudorabies) in pigs. In: Wittmann G
(ed) Herpesvirus diseases of cattle, horses, and pigs. Kluwer Academic Publishers,
Boston, pp 230325
3. Pensaert MB, Kluge JP (1989) Pseudorabies virus (Aujeszky's disease). In: Pensaert
MB (ed) Virus infections of porcines. Elsevier Scientific Publishers, Amsterdam,
4. Kluge JP, Beran GW, Hill HT, Platt KB (1992) Pseudorabies (Aujeszky's disease). In:
Leman AD, Straw B, Mengeling WL, D'Allaire S, Taylor DJ (eds) Diseases of swine,
7th edn. Iowa State University Press, Ames, pp 312-323
5. Wittmann G (1991) Spread and control of Aujeszky's disease (AD). Comp Immunol
Microbiol Infect Dis 14:165-173
6. Vandeputte J, Chappuis G, Fargeaud D, Precausta P, Guillemin F, Brun A, Desmettre
Ph, Stellmann C (1990) Vaccination against pseudorabies with glycoprotein gI + or
glycoprotein gI- vaccine. Am J Vet Res 51:1100-1106
7. Van Oirschot JT, De Waal CAH (1987) An ELISA to distinguish between Aujeszky's
disease vaccinated and infected pigs. Vet Rec 121:305-306
8. Wardley RC, Post LE (1989) The use ofgX deleted vaccine PRV TK gX-1 in the control
of Aujeszky's disease. In: Van Oirschot JT (ed) Vaccination and control of Aujeszky's
disease. Kluwer, Dordrecht Brussel, pp 13-25
9. Mengeling WL, Lager KM, Brockmeier SL (1992) Effect of various vaccination
procedures on shedding, latency, and reactivation of attenuated and virulent pseudorabies
virus in swine. Am J Vet Res 53:2164-2173
10. Tartaglia J, Perkus ME, Taylor J, Norton EK, Audonnet JC, Cox WI, Davis SW, van
der Hoeven J, Meignier B, Riviere M, Languet B, Paoletti E (1992) NYVAC: A highly
attenuated strain of vaccinia virus. Virology 188:217-232
11. Brockmeier SL, Lager KM, J Tartaglia J, Riviere M, Paoletti E, Mengeling WL (1993)
Vaccination of pigs against pseudorabies with highly attenuated vaccinia (NYVAC)
recombinant viruses. Vet Microbiol 38:41-58
12. Mengeling WL (1991) Virus reactivation in pigs latently infected with a thymidine
kinase-negative vaccine strain of pseudorabies virus. Arch Virol 120:57 70
13. Mengeling WL, Van Der Maeten MJ (1988) Preparation of bovine Embryonic spleen
celt cultures. J Tissue Cult Methods 11:135-138
14. Marchioli CC, Yancy Jr RV, Wardley RC, Thomsen DR, Post LE (1987) A vaccine
strain of pseudorabies virus with deletions in the thymidine kinase and glycoprotein X
genes. Am J Vet Res 48:1577-1583
Vaccinia-virus-vectored vaccine for pseudorabies 269
15. Riviere M, Tartaglia J, Perkus ME, Norton EK, Bongermino CM, Lacoste F, Duret
C, Desmettre P, Paoletti (1992) Protection of mice and swine from pseudorabies virus
conferred by vaccinia virus-based recombinants. J Virol 66:3424-3434
Authors' address: Dr. W. L. Mengeling, USDA, ARS, NADC, 2300 Dayton Avenue,
P.O. Box 70, Ames, IA 50010, U.S.A.
Received July 15, 1993