IE83654B1 - Bovine herpesvirus type I deletion mutants, vaccines based thereon, diagnostic kits for detection of bovine herpesvirus type I - Google Patents
Bovine herpesvirus type I deletion mutants, vaccines based thereon, diagnostic kits for detection of bovine herpesvirus type IInfo
- Publication number
- IE83654B1 IE83654B1 IE1992/2829A IE922829A IE83654B1 IE 83654 B1 IE83654 B1 IE 83654B1 IE 1992/2829 A IE1992/2829 A IE 1992/2829A IE 922829 A IE922829 A IE 922829A IE 83654 B1 IE83654 B1 IE 83654B1
- Authority
- IE
- Ireland
- Prior art keywords
- bhv
- gene
- deletion
- specific
- protein
- Prior art date
Links
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Description
Title: Bovine herpesvirus type 1 deletion mutants,
vaccines
based thereon, diagnostic kits for detection of bovine
herpesvirus type 1.
FIELD OF THE INVENTION
This invention relates to the fields of vaccination and
diagnostics in connection with diseases which are caused by
pathogens and involves the use of both the classic methods to
arrive at a live attenuated vaccine or an inactivated vaccine
and the modern methods based on DNA recombinant technology.
More specifically, the invention relates to live
attenuated vaccines and inactivated vaccines for protecting
animals, especially bovines,
(BHV-1),
against bovine herpesvirus type l
these vaccines being so designed that they are not
only safe and effective, but also create the possibility of
distinguishing infected from non-infected animals in a
vaccinated population.
Diagnostic kits which can be used for such a test for
distinguishing infected from non-infected animals in a
vaccinated population are also an aspect of the present
invention.
BACKGROUND OF THE INVENTION
BHV-1,
(IBRV)
including the infectious bovine rhinotracheitis
virus and the infectious pustular vulvovaginitis virus
(IPVV), plays an important role in the development of
respiratory diseases and fertility disorders in bovines. After
an acute infection, BHV-1 often remains present in the host in
a latent form. Latent virus can be reactivated under the
influence of inter alia stress - which may or may not be
accompanied by clinical phenomena — and subsequently excreted.
As a consequence, infected cattle must be regarded as lifelong
potential spreaders of BHV-1. BHV-1 occurs endemically in an
estimated 75% of Dutch cattle farms. Especially older cattle
are serologically positive.
‘83654
There are a number of inactivated ("dead") vaccines and a
("live")
inoculation against BHV-1 infections.
number of attenuated vaccines available for
Inactivated vaccines are
prepared by killing the BHV-1 virus, for instance by heat
treatment, irradiation or treatment with ethanol or formalin.
However, these often give insufficient protection. Attenuated
vaccines are prepared by a large number of passages on
homologous (bovine) or on heterologous cells such as porcine
or canine cells, and sometimes viruses are also treated
physically or chemically then. In this way, unknown
mutations/deletions develop in the virus genome, which often
reduce the disease-producing properties of the virus.
Attenuated live vaccines give better protection than
inactivated vaccines, inter alia because they present more
viral antigens to the immune system of the host. Another
important advantage of live vaccines is that they can be
administered intranasally, i.e., at the site where the first
multiplication of the wild type virus occurs after infection.
Yet, Some live
live vaccines leave room for improvement.
vaccines still seem to possess their abortogenic ability,
which becomes manifest in particular after intramuscular
administration. Moreover, probably all live vaccines remain
latently present in the vaccinated cow. Also, there is a
chance that if the vaccine differs only little from the wild-
type virus, But one of the
major problems is that the BHV-1 vaccines cannot prevent
reversion to virulence will occur.
infection by wild-type viruses. The result is that vaccinated
cattle can also spread wild-type BHV-1.
For a proper BHV-1 control program, it is necessary to
have disposal of an efficacious and safe vaccine that can be
distinguished from wild-type virus, since the application of
an efficacious vaccine can reduce the circulation of BHV-1
considerably and a test which can distinguish between a
vaccine and a wild-type virus makes it possible to detect (and
then remove) infected cattle in a vaccinated population.
Meanwhile, BHV—l vaccines have been developed which seem
to be safer than conventional vaccines and are distinguishable
from wild-type virus. A thymidine kinase deletion mutant has
been isolated which is abortogenic to a lesser degree, becomes
latent less frequently and cannot be reactivated. Further,
using recombinant DNA techniques, a BHV—l vaccine has been
constructed which has a deletion in the gene for glycoprotein
gIII, which makes this vaccine distinguishable from wild-type
BHV-1 by means of serological techniques. However, there are
still some objections to these vaccines. On the one hand, the
thymidine kinase gene is involved in the viral replication and
less replication can lead to less protection. On the other
hand, the glycoprotein glII is important for generating
protective antibodies, which makes a gIII deletion vaccine
less effective. A practical problem is that intranasal
administration, which generally gives the best protection, of
recombinant vaccines is not allowed in some countries.
Accordingly, there is a need for a vaccine which is safe as
well as effective and yet can be distinguished from wild-type
BHV—l, it being further desirable that at least one of such
vaccines is based on a virus attenuated via a conventional
route rather than a virus constructed by recombinant DNA
techniques.
Now, via passages in cell cultures, a BHV—l strain has
been obtained which lacks the gene for glycoprotein gE. The
first results of our research indicate that this gene is quite
useful to make a serological distinction with regard to wild-
type BHV—l and that it is involved in the expression of
virulence. Therefore, its deletion contributes to safety and
may render the use of thymidine kinase deletions superfluous.
The glycoprotein gE seems to be less important for induction
of protection than the glycoprotein gIII. A conventionally
attenuated BHV—l strain which can be serologically
distinguished from wild~type virus is unique. The location and
DNA sequence of the gE gene described herein for the first
time were not previously known, nor were oligonucleotides,
polypeptides and oligopeptides that can be derived therefrom.
A test for making a serological distinction on the basis of
the gE gene is also unique.
An important advantage of this "conventional" gE deletion
("conventional" refers to the use of a conventional
is that it will be
possible to administer it intranasally in countries where this
mutant
method for isolating an attenuated virus)
is forbidden as far as recombinant vaccines are concerned.
Taking due account of the different views on safety, however,
well-
defined recombinant versions have been constructed as well.
in addition to this conventional gE deletion vaccine,
These recombinant vaccines also have a gE deletion - and may
or may not have a deletion in the thymidine kinase gene as
well - and can also be used as vectors for the expression of
heterologous genes. All these recombinant vaccines can be
distinguished from wild-type virus with the same gE-specific
test. The use of a standard test for a set of different
vaccines can be a great advantage in the combat of BHV—l as an
international effort. Such an approach has not been previously
described in the field of BHV—l vaccines.
Serological analysis of the anti BHV-1 response in cattle
showed that an important fraction of the anti—gE antibodies
are directed against a complex formed by glycoprotein gE and
another BHV-1 glycoprotein: glycoprotein gl. Serological tests
that can (also) demonstrate the presence of such complex-
specific antibodies may therefore be more sensitive than tests
that can only detect anti—gE antibodies. Cattle vaccinated
with a single gE deletion mutant may produce anti-gI
antibodies that can interfere with the detection of anti-gI/gE
antibodies. Consequently, this invention also includes a
vaccine with a gI/gE double deletion.
SUMMARY OF THE INVENTION
In the first place, this invention provides a deletion
mutant of BHV—1 which has a deletion in the glycoprotein gE—
gene. The words "a deletion in" intend to cover a deletion of
the gene as a whole.
A preferred embodiment of the invention is constituted by
a deletion mutant of BHV—1 which has a deletion in the
glycoprotein gE-gene which has been caused by an attenuation
procedure, such as the deletion mutant Difivac-1 to be
described hereinafter.
Other preferred embodiments of the invention consist of a
deletion mutant of BHV—1 comprising a deletion in the
glycoprotein gE—gene which has been constructed by recombinant
DNA techniques, such as the deletion mutants 1B7 or 1B8 to be
described hereinafter.
Another preferred embodiment of the invention consists of
a double deletion mutant of BHV—1 comprising a deletion in the
glycoprotein gE-gene and a deletion in the glycoprotein gl-
gene, such as the gI/gE double deletion mutant Difivac—IE to
be described hereinafter.
Further, with a view to maximum safety, according to the
invention a deletion mutant of BHV—1 is preferred which has a
deletion in the glycoprotein gE-gene and a deletion in the
thymidine kinase gene. The invention also covers a deletion
mutant of BHV—1 which has a deletion in the glycoprotein gE-
gene, the glycoprotein gI—gene and the thymidine kinase gene.
The invention provides a vaccine composition for
vaccination of animals, in particular mammals, more
particularly bovines, to protect them against BHV—1,
comprising a deletion mutant of BHV—1 as defined hereinabove,
and a suitable carrier or adjuvant. Said composition may be a
live or an inactivated vaccine composition.
The invention is further embodied in a mutant of BHV—1
which has a deletion in the glycoprotein gE-gene and contains
a heterologous gene introduced by recombinant DNA techniques.
Preferably, this concerns a mutant of BHV-1 which contains a
heterologous gene introduced by recombinant DNA techniques at
the location of the glycoprotein gE—gene, which heterologous
gene is under the control of regulatory sequences of the gE-
gene and is optionally attached to the part of the gE—gene
which codes for a signal peptide. Said heterologous gene may
also be under the control of a different promoter of BHV-1, or
under the control of a heterologous promoter. When the mutant
of BHV-1 has further deletions in addition to the deletion in
the glycoprotein gE-gene, such as a deletion in the thymidine
kinase gene and/or a deletion in the glycoprotein gI-gene,
said heterologous gene may also be inserted at the location of
this additional deletion(s). Plural insertions are another
option, either together at the location of one deletion, or
distributed over locations of several deletions.
The heterologous gene introduced preferably codes for an
immunogenic protein or peptide of another pathogen, or for a
cytokine which promotes the immune response. Examples of
suitable cytokines are interleukin 2, interferon-alpha and
interferon-gamma.
The invention also provides a (live or inactivated)
vaccine composition for vaccination of animals, in particular
mammals, more particularly bovines, to protect them against a
(different) pathogen, comprising a mutant of BHV-l having
therein a heterologous gene coding for an immunogenic protein
or peptide of that other pathogen, and a suitable carrier of
adjuvant. Of course, the protection may concern more than one
pathogen, i.e. a multivalent vaccine wherein the mutant
contains a plurality of heterologous genes.
The invention further relates to a composition comprising
a recombinant nucleic acid comprising the glycoprotein gE—gene
of BHV-l, a part of this glycoprotein gE—gene or a nucleotide
sequence derived from this glycoprotein gE-gene. This
composition can contain a cloning or expression vector having
therein an insertion of a recombinant nucleic acid which
comprises the glycoprotein gE—gene of BHV—l, a part of this
glycoprotein gE-gene or a nucleotide sequence derived from
this glycoprotein gE-gene.
The invention also comprises a composition comprising
glycoprotein gE of BHV—l, a part of this glycoprotein gE, a
peptide derived from this glycoprotein gE, or a complex of the
glycoproteins gE and g1, and a composition comprising an
antibody which is specific for glycoprotein gE of BHV-1, a
part of this glycoprotein gE, a peptide derived from this
glycoprotein gE, or a complex of the glycoproteins gE and g1.
"Antibody" is understood to mean both a polyclonal antibody
preparation and a monoclonal antibody preferred for most
applications. The terms "a part of glycoprotein gE" and "a
peptide derived from glycoprotein gE" are understood to mean
gE-specific amino acid sequences which generally will have a
length of at least about 8 amino acids.
The invention further relates to a diagnostic kit for
detecting nucleic acid of BHV-1 in a sample, in particular a
blood cells,
lung lavage fluid, nasal
biological sample such as blood or blood serum,
milk, bodily fluids such as tears,
fluid,
tissue,
sperm, in particular seminal fluid, saliva, sputum, or
in particular nervous tissue, coming from an animal,
particularly a mammal, more particularly a bovine, comprising
a nucleic acid probe or primer having a nucleotide sequence
derived from the glycoprotein gE-gene of BHV—1, and a
detection means suitable for a nucleic acid detection assay.
Further, the invention relates to a diagnostic kit for
detecting antibodies which are specific for BHV—1, in a
sample, in particular a biological sample such as blood or
blood serum, saliva, sputum, bodily fluid such as tears, lung
lavage fluid, nasal fluid, milk, or tissue, coming from an
animal, in particular a mammal, more in particular a bovine,
comprising glycoprotein gE of BHV—l, a part of this
glycoprotein gE, a peptide derived from this glycoprotein gE,
or a complex of the glycoproteins gE and gI, and a detection
means suitable for an antibody detection assay. Such a
diagnostic kit may further comprise one or more antibodies
which are specific for glycoprotein gE of BHV-1 or specific
for a complex of the glycoproteins gE and gI of BHV-1.
The invention also relates to a diagnostic kit for
detecting protein of BHV—l in a sample, in particular a
blood cells,
lung lavage fluid, nasal
biological sample such as blood or blood serum,
milk, bodily fluids such as tears,
fluid, sperm, saliva,
in particular seminal fluid, sputum or
tissue, in particular nervous tissue, coming from an animal,
in particular a mammal, more in particular a bovine,
comprising one or more antibodies which are specific for
glycoprotein gE of BHV-1 or specific for a complex of the
glycoproteins gE and g1 of BHV—1, and a detection means
suitable for a protein detection assay.
The invention further provides a method for determining
BHV—1 infection of an animal, in particular a mammal, more in
particular a bovine, comprising examining a sample coming from
the animal, in particular a biological sample such as blood or
blood cells,
sputum, bodily fluid such as tears,
blood serum, sperm, in particular seminal fluid,
saliva, lung lavage fluid,
nasal fluid, milk, or tissue, in particular nervous tissue,
for the presence of nucleic acid comprising the glycoprotein
gE-gene of BHV-1, or the presence of the glycoprotein gE of
BHV—l or a complex of the glycoproteins gE and g1 of BHV—l, or
the presence of antibodies which are specific for the
glycoprotein gE of BHV—l or specific for a complex of the
glycoproteins gE and gI of BHV-1. The sample to be examined
can come from an animal which has not been previously
vaccinated with a vaccine composition according to the
invention or from an animal which has previously been
vaccinated with a vaccine preparation according to the
invention.
DETAILED DESCRIPTION OF THE INVENTION
The invention relates to a set of BHV—1 vaccines, both
live and inactivated, which have in common that they lack the
glycoprotein gE gene in whole or in part. This set comprises
both a natural gE deletion mutant and constructed gE deletion
mutants which may or may not also comprise a deletion of the
thymidine kinase gene and/or the glycoprotein gI gene, and
constructed gE deletion mutants which are used as vectors for
heterologous genes. The invention further relates to
nucleotide sequences encoding the BHV—1 glycoprotein gE—gene,
the
glycoprotein gE itself, peptides which are derived therefrom
oligonucleotides derived from these sequences,
and (monoclonal or polyclonal) antibodies which are directed
against the gE glycoprotein and peptides derived therefrom.
The invention also relates to complexes of the glycoproteins
gE and gI of BHV-l, and to antibodies directed against such
complexes.
These materials according to the invention can be used
for:
1) the vaccination of cattle against diseases caused by
BHV-l, such that a distinction can be made between BHV—1
infected animals and vaccinated animals; the conventional and
the constructed vaccine can be used side by side;
) the vaccination of cattle against both BHV-1 diseases and
diseases caused by other pathogens of which coding sequences
for protective antigens can be incorporated into the BHV—1
deletion mutants;
3) testing blood, serum, milk or other bodily fluids from
cattle to determine serologically or by means of nucleic acid
(e g. PCR)
infected by a wild-type BHV-l or have been vaccinated with a
detection techniques whether they have been
gE deletion mutant.
Synthesis of oligopeptides, polypeptides and glycoproteins
derived from the coding sequence of the glycoprotein gE-gene
and the glycoprotein gI-gene of BHV—1
described in
(Fig. 3A) and the
isolated DNA fragments which code for this gene, make it
The results of the DNA sequence analysis,
the examples, of the glycoprotein gE-gene
possible, using standard molecular-biological procedures, both
to synthesize peptides of the gE protein (oligo or
polypeptides) and to express the gE protein in its entirety or
in large parts via the prokaryotic route (in bacteria) or via
the eukaryotic route Via these
(for instance in murine cells).
routes, gE—specific antigen can be obtained which can for
instance serve for generating gE—specific monoclonal
(Mabs).
specific Mabs)
antibodies Furthermore, gE-specific antigen (and gE—
can be used in serological tests to enable a
distinction to be made between animals vaccinated with a BHV—1
gE deletion vaccine and animals infected with wild-type BHV—1
virus.
The results of the partial DNA sequence analysis of the
glycoprotein gI gene - described in the examples — and the
isolated DNA fragments that code for this gene, together with
the eukaryotic cells expressing glycoprotein gE, allow the
expression of the gI/gE complex in eukaryotic cells (See
Figures 13 and 14). This glycoprotein complex can be used to
produce gI/gE specific monoclonal antibodies. The gI/gE
complex can also be used as antigen in serological tests to
differentiate between cattle vaccinated with a single gE BHV—1
deletion mutant or with a double gI/gE BHV—1 deletion mutant
and cattle infected with wild type BHV—1 virus.
gE-specific peptides
On the basis of a known protein coding sequence, by means
of an automatic synthesizer, polypeptides of no less than
about 40-50 amino acids can be made. Now that the protein
coding sequence of the gE glycoprotein of BHV—1 strain Lam has
been unraveled (Fig. 3A), polypeptides of this BHV—1 gE
glycoprotein can be synthesized. With such polypeptides,
according to standard methods, experimental animals such as
mice or rabbits can be immunized to generate gE—specific
antibodies. Further, using these gE—specific peptides, the
locations where anti—gE antibodies react with the gE protein
(the epitopes)
PEPSCAN method
USA 81,
can be further specified, for instance with the
1984, Natl. Acad. Sci.
gE-specific oligopeptides can also be used
(Geysen et al.,
3998-4002).
in serological tests which demonstrate anti-gE antibodies.
Proc.
Prokaryotic expression of gE
For the synthesis of the gE protein in bacteria (i.e. the
prokaryotic expression of gE), DNA fragments which code for
the glycoprotein gE or for parts thereof must be cloned into
prokaryotic expression vectors. Prokaryotic expression vectors
are circular DNA molecules which can maintain themselves in a
bacterium as a separately replicating molecule (plasmid).
These expression vectors contain one or more marker genes
which code for an antibiotic resistance and thus enable the
selection for bacteria with the expression vector. Further,
expression vectors comprise a (often controllable) promoter
region behind which DNA fragments can be ligated which are
then expressed under the influence of the promoter. In many
current prokaryotic expression vectors, the desired protein is
expressed while fused to a so-called carrier protein. To that
end, in the vector there is located behind the promoter the
coding sequence for the carrier protein, directly adjacent to
which the desired DNA fragment can be ligated. Fusion proteins
are often more stable and easier to recognize and/or to
isolate. The steady-state level which a particular fusion
protein can attain in a certain bacterial strain differs from
fusion to fusion and from strain to strain. It is customary to
try different combinations.
Eukaryotic expression of the glycoprotein gE—gene
Although prokaryotic expression of proteins offers some
advantages, the proteins lack the modifications, such as
glycosylation and the like, which occur in eukaryotic cells.
As a result, eukaryotically expressed protein is often a more
suitable antigen. For the heterologous expression of proteins
in eukaryotic cells, use is made of
such as murine cells,
eukaryotic expression vectors. These vectors are plasmids
which can not only be multiplied in E. coli cells but also
subsist stably in eukaryotic cells. In addition to a
prokaryotic selection marker, they also comprise a eukaryotic
selection marker. Analogously to the prokaryotic expression
vectors, eukaryotic expression vectors contain a promoter
region behind which desired genes can be ligated. However, the
promoter sequences in eukaryotic vectors are specific for
eukaryotic cells. Moreover, in eukaryotic vectors fusion to
carrier proteins is utilized only rarely. These vectors are
introduced into the eukaryotic cells by means of a standard
1973,
In addition to the eukaryotic plasmid
transfection method (F.L. Graham and A.J.
456-467).
there are also viral vectors,
van der Eb,
Virology 52,
vectors, where the heterologous
gene is introduced into the genome of a virus (e.g.
retroviruses, herpesviruses and vaccinia virus). Eukaryotic
cells can then be infected with recombinant viruses.
In general, it cannot be predicted what vector and cell
type are most suitable for a particular gene product. Mostly,
several combinations are tried.
Eukaryotic expression of both the glycoprotein gE and the
glycoprotein gI
The final structure that a protein obtains, is depending
on its primary amino acid sequence, its folding, its
posttranslational modifications etc. An important factor that
contributes to structure of a protein is its interaction with
one or more other proteins. We have found that also BHV—l
glycoprotein gE forms a complex with at least one other
glycoprotein: BHV—1 glycoprotein gI. The first indication for
such a complex came from our results with candidate anti—gE
Mabs 1, 51, 67, 75 and 78 (See table 2).
react with Difivac—l, nor with Lam gE‘ but also failed to
These Mabs did not
recognize glycoprotein gE—expressing 3T3 cells. However, these
Mabs did react with gE-expressing 3T3 cells after infection
with Difivac-1, showing that complementing factors are needed
to give glycoprotein gE the proper antigenic conformation for
these Mabs. In some of our radio-immunoprecipitation
experiments with Mab 81 we found coprecipitation of a protein
with an apparent molecular weight of 63 kD. In view of the
fact that the herpes simplex virus glycoprotein gE forms a
complex with a protein with a comparable molecular weight
(HSV1 glycoprotein gI), we inferred that BHV-l glycoprotein gE
forms a complex with the BHV-l homolog of glycoprotein gI. To
study this BHV—1 gE/gI complex and to produce gE antigen with
the proper antigenic structure we expressed both glycoproteins
in one eukaryotic cell. For this we applied the same
procedures as described for the eukaryotic expression of
glycoprotein gE alone. The only additional prerequisite is the
use of expression vectors with different eukaryotic selectable
markers.
Serological tests
Serological methods for making a distinction between
cattle vaccinated with Difivac—l and cattle infected with
wild-type BHV—1 on the basis of antibodies against gE are
preferably based on the use of monoclonal antibodies directed
against gE. These can be used in the following manners:
a) According to the principle described by Van Oirschot et
191-206, 1988). In
this ELISA for the detection of gI antibodies against the
al. (Journal of Virological Methods 22,
virus of Aujeszky‘s disease, antibodies are demonstrated by
their blocking effect on the reaction of two Mabs having two
different epitopes on gI. The test is carried out as follows.
Microtiter plates are coated with Mab 1, overnight at 37°C,
after which they are stored, e.g. at 4°C or -20°C. The serum
to be examined is preincubated with antigen in separate
e.g. for 2 h at 37°C. The Mab l-
e.g. 5 times, after which Mab 2
(HRPO) is added to these
plates. Then the preincubated serum—antigen mixtures are
uncoated microtiter plates,
coated plates are washed,
coupled to horseradish peroxidase
transferred to the plates in which the two Mabs are located,
followed by incubation, e.g. for l h at 37°C. The plates are
washed and substrate is added to each well. After e.g. 2 h at
room temperature, the plates are spectrophotometrically read.
Four negative control sera and four serial dilutions of a
positive serum are included on each plate. The serum which has
an optical density (OD) value of less than 50% of the average
OD value of the 4 negative control sera which have been
examined on the same plate, is considered positive.
b) According to the Indirect Double Antibody Sandwich (IDAS)
principle. Here, microtiter plates are coated with an Mab or a
polyclonal serum directed against the gE protein. Incubation
with a gE-antigen preparation results in gE binding to the
coating. Antibodies specifically directed against gE in the
bovine serum to be examined subsequently bind to the gE. These
bound antibodies are recognized by an anti—bovine
immunoglobulin conjugate. The antibodies in this conjugate are
the bound
conjugate is visualized by addition of a chromogenic
covalently bound to peroxidase enzyme. Finally,
substrate. The specificity of the reaction is checked by
carrying out the same procedure with a gE-negative control
preparation instead of a gE-antigen preparation. On each
microtiter plate, positive and negative control sera are
included. The test is valid if the positive serum scores
positive in a certain dilution. A serum is positive if it
scores an OD which is 0.2 higher than the standard negative
control serum.
c) According to the IDAS principle as described under 2, but
after incubation of the serum to be examined an anti-gE
Mab/HRPO is used instead of the anti-bovine immunoglobulin
conjugate. An anti—gE peptide serum or an anti—gE polyclonal
serum may be used instead of the anti—gE Mab. The plates are
washed and to each well a chromogenic substrate is added.
After e.g. 2 h at room temperature, the plates are
spectrophotometrically read. Four negative control sera and
four serial dilutions of a positive serum are included on each
plate. The serum which has an OD value of less than 50% of the
average OD value of the 4 negative control sera which have
been examined on the same plate, is considered positive.
d) According to the principle of a blocking ELISA, whereby
virus antigen which may or may not be purified is coated to
the microtiter plate overnight.
In these plates, the serum to
be examined is incubated for, e.g. one hour or longer at 37°C.
After a washing procedure, an anti—gE Mab is added to the
l h at 37°C. An anti-
gE peptide serum or an anti—gE polyclonal serum may be used
plates, followed by incubation for e.g.
instead of the anti—gE Mab. The plates are washed and to each
well a chromogenic substrate is added. After e.g. 2 h at room
temperature, the plates are read spectrophotometrically. Four
negative control sera and four serial dilutions of a positive
serum are included on each plate. The serum which has an OD
value of less than 50% of the average OD value of the 4
negative control sera which have been examined on the same
plate, is considered positive.
In all the above arrangements, conventionally grown virus
antigen which contains gE can be used, but so can gE-antigen
which is expressed via prokaryotes or eukaryotes.
Alternatively, oligopeptides based on the BHV-1 gE sequence
could be used in the above diagnostic tests instead of
conventional antigen. In addition, such oligopeptides could be
used for the development of a so—called "cow-side" test
according to the principle described in an article by Kemp et
1352-1354, 1988.
based on a binding of the antigenic sequence of the
al., Science 241, Such a test would then be
oligopeptide by antibodies directed against gE, present in
infected animals. For such a test, the oligopeptide would have
to be coupled to an Mab directed against bovine erythrocytes.
Nucleic acid analysis using the polymerase chain reaction
Oligonucleotides (probes and primers) can for instance be
used in the polymerase chain reaction to make a distinction
between vaccinated and infected animals.
(PCR)
The polymerase chain
reaction is a technique whereby nucleic acids of a
pathogen can be multiplied billions of times in a short time
(De polymerase kettingreactie, P.F. Hilderink, J.A. Wagenaar,
1990,
1111-1117). The gE
oligonucleotides can be chosen such that in a gE positive
J.W.B. van der Giessen and B.A.M. van der Zeijst,
Tijdschrift voor Diergeneeskunde deel 115,
genome a different product is formed than in a gE negative
genome. The advantage of this is that also an animal which has
been vaccinated with a gE deletion vaccine gives a positive
signal in a PCR test. However, this approach depends on the
presence of nucleic acids of the virus in a sample, for
instance blood, coming from the animal to be tested.
After an acute BHV—l infection, there is a great chance
that BHV-l specific nucleic acids can be demonstrated in the
blood, but it has not been determined yet whether BHV-1
nucleic acids can also be demonstrated in the blood during
latency.
The use of BHV—l as a vector
For expressing heterologous genes in the BHV—l genome, it
is necessary to have disposal of exact information on the area
where the heterologous gene is to be inserted. There should
not be any disturbance of essential sequences, and regulatory
sequences must be available for the expression of the
heterologous gene. In principle, the glycoprotein gE-gene is a
suitable place to express heterologous genes. The gE—gene is
not essential, hence there is no objection to replacing the gE
the
heterologous gene can be so positioned that it will be under
gene by the heterologous gene. As a consequence,
the influence of the regulatory sequences of the gE gene.
However, it is not necessary to use the regulatory sequences
of the gE-gene. The expression of heterologous genes may be
controlled alternatively by other,
e.g. stronger regulatory
sequences of different genes. It is also possible to ligate
the heterologous gene to the (export) signal peptide of the gE
gene, so that the secretion of the heterologous gene product
can be influenced. It is clear that detailed knowledge of the
gE gene and the gE protein affords the possibility of using
BHV-1 as a vector in a very measured manner. The vectors
developed can moreover be serologically distinguished from
wild—type. The construction of BHV—l mutants which express
heterologous genes can be carried out in the same manner as
the construction of gE deletion mutants shown in the examples.
However, the deletion fragments should then be replaced with a
fragment on which a heterologous gene is located at the
location of the deletion.
EXAMPLES
l) Isolation and identification of a natural gE deletion
mutant
a) Isolation of a natural mutant
Genomic DNA was isolated from a number of conventionally
attenuated vaccines according to standard methods and analyzed
using restriction enzymes. In particular, we searched for
genome deviations which would be suitable to enable
distinction from wild—type BHV—l virus.
Attention was directed in particular to the Us region of
the BHV—1 genome, because in that region — by analogy with the
herpes simplex virus ~ probably a number of genes coding for
non—essential glycoproteins are located [Identification of a
herpes simplex virus I glycoprotein gene within a gene cluster
dispensable for growth in cell culture, R. Longnecker, S.
Chatterjee, R.J. Whitley and B. Roizman (1987) Proc. Natl.
Acad. Sci. 84, 4303-4307].
A batch of a BHV-l vaccine coming from the University of
Zagreb, Yugoslavia
241-245, 1985),
embryonal kidney cells and embryonal bovine trachea cells
(Lugovic et al., Veterinarski Arhiv 55,
after a great number of passages on bovine
(Ebtr), proved to have a deviant Us region in addition to a
normal Us region. This vaccine moreover appeared to form both
large and small plaques on Ebtr cells. From this mixed
population, a virus with a deviant Us region was isolated by
three limiting dilution steps, with small plaques being chosen
each time. The virus isolated via this route was examined
further and called Difivac—l. It was deposited with Institut
Pasteur, on 27 May 1992, deposit number I—l2l3.
Paris, France,
b) Identification of the deletion in the gE gene in
Difivac-l
For further analysis of this deviation in the Us region,
genomic DNA of Difivac-l was isolated according to standard
(Fig. 1A).
Hybridization of this blot with a 32P labeled wild-type
methods and subjected to Southern blot analysis
HindIII K fragment confirmed that this fragment, located
(kb)
in Difivac-l. Moreover, by this analysis, the position of the
1B).
analysis of this deletion, the Us region of the wild-type
centrally in the Us region, is some 1.0 kilobase shorter
missing part could be approximated (Fig. For further
BHV—l strain Lam was isolated and cloned into prokaryotic
To that end,
DNA of the Lam strain (Fig. 2A) was isolated and cloned into
the vectors pUCl8, pACYC and pBR322
of the area around the supposed position of the deletion was
composed (Fig. 2C). Starting from this physical map,
suitable for the determination of the nucleotide sequence of
vectors. according to standard methods, genomic
(Fig. 2B). A physical map
subclones
this area were constructed in the vectors pKUNl9 and pUCl8
(Fig. 2D). Using these subclones, the nucleotide sequence of
the two strands of the entire area
(indicated in Fig. 2C) was
determined using the Sanger method. This nucleotide sequence
(SEQ ID NO:l)
conceptual translation,
was analyzed using the PC/Gene program. From the
(nt) 168
through nt 1893 code for an open reading frame of 575 amino
it appeared that nucleotides
acids (Fig. 3A). Further analysis showed that this amino acid
sequence has the characteristics of a transmembrane
glycoprotein as is shown in Fig. 3B. The fact is that the
first 26 amino acids (aa) are recognized as a typically
eukaryotic export signal and the area between aa 423 and aa
450 is recognized as a transmembrane region. In addition,
three potential N—bound glycosylation sites occur in this
sequence. This predicted amino acid sequence exhibits clear
similarities to the glycoprotein gE-gene of herpes simplex
virus (HSV); see Figs 4A and 4B. These and other similarities
justify the conclusion that the gene found is the gE homologue
of BHV—l. For this reason, the gene is called gE. To determine
to what extent this BHV—1 gE-gene is missing in Difivac-1, the
p318 fragment was isolated. The p318 fragment starts on the
AluI site 55 nt before the postulated BHV—1 gE open reading
frame and ends 133 nt behind it. Genomic Difivac—l DNA was
analyzed with this p318 fragment using Southern blot
hybridization. This revealed that Difivac—l contains no p318
detectable sequences (Fig. 5). This experiment confirmed that
Difivac-1 contains a deletion and clearly demonstrates that
this deletion extends throughout the entire gE gene.
To determine the size and the position of the deleted
region, genomic sequences covering the Us region of Difivac—l
were cloned into prokaryotic vectors. See Figure 11C. The
14.5 kb EcoRI fragment was cloned into the pACYC vector and
named p775. The 7.4 kb HindIII fragment was independently
cloned into the pUC18 vector and named p728. From clone p728
two subclones were isolated: the 1.4 kb PstI fragment in clone
p737 and the 350bp AluI-PstI fragment in clone p754.
Restriction enzyme analysis and Southern blot analysis of
demonstrated that the gE
deletion in Difivac—l is 2.7 kb long,
these clones (data not shown),
starting just 5' from
the gE gene and ending at the border of the Us region. These
2.7 kb have been replaced by a duplication of a 1 kb segment,
located in the Us region opposite to the gE gene, as an
aberrant extension of the repeat region. See figure llB. To
confirm the results of this analysis and to determine the
exact recombination point, the nucleotide sequence of most of
the insert of clone p754 was determined and compared with the
wild type sequences. See figure l2. This analysis showed that
the recombination point is located 77 bp upstream from the
start codon of the gE gene.
c) Evaluation of safety and efficacy of Difivac—l
Difivac-l was tested in BHV—l seronegative specific
pathogen free calves of seven week old. Eight calves were
intranasally vaccinated with 105 TCID5o in 2 ml, of which 1 ml
was sprayed in each nostril. Eight BHV—l seronegative specific
pathogen free calves of seven week old, that were housed in a
separate isolation unit, were given 2 ml of culture medium
intranasally,
and served as unvaccinated controls. Five weeks
after vaccination, vaccinated and control calves were
challenged intranasally with 107 TCID5o of the highly virulent
BHV—l strain Iowa. Six weeks after challenge all the calves
were treated intramuscularly with dexamethasone for 5 days to
reactivate putative latent virus. Clinical signs, rectal
temperatures and body growth were monitored. Virus isolations
were performed from nasal swabs, and neutralizing antibody
titres were determined in serum.
After vaccination, rectal
behaviour, appetite,
temperatures and growth rates of the calves remained normal,
but the vaccinated calves had some serous nasal discharge and
some hypersalivation. Lesions in nasal mucosa were not
observed. Difivac—l was excreted from nasal swabs after
vaccination (Fig. 17). All vaccinated calves produced
neutralizing antibodies to BHV-l.
After challenge, all unvaccinated control calves showed
apathy, loss of appetite, ocular and nasal discharge,
reddening of the gingiva of the lower jaw, severe lesions of
the nasal mucosae until 14 days after challenge, and a growth
arrest of 4 days. The vaccinated calves had small, quickly
healing lesions of the nasal mucosae and had no growth arrest.
The daily clinical scores, the rectal temperature and growth
19 and 20.
but
the amount and period of virus excretion was markedly reduced
development after challenge are given in Figs. l8,
After challenge, all calves shed virus from their nose,
in vaccinated calves (Fig. 21). A secondary antibody response
developed in vaccinated calves and the unvaccinated calves all
produced antibodies after challenge.
After reactivation, the challenge virus was isolated from
one vaccinated calf and from 5 unvaccinated calves. Difivac-l
could not be reactivated.
The above results demonstrate that Difivac-l hardly
induced any sign of disease in young calves and was not
reactivatable. Difivac-l markedly reduced the severity of
disease and the amount of virus excretion after challenge.
In conclusion, Difivac-l is a safe and efficacious
vaccine for use in cattle against BHV-1 infections.
) Construction of recombinant gE deletion mutants of BHV-1
In order to be able to have disposal of differentiatablc
BHV—1 vaccines which are molecularly better defined than
Difivac-l and which,
if so desired, contain a deletion in for
instance the thymidine kinase gene, in addition to a deletion
in the gE gene, recombinant gE deletion mutants were
constructed, in addition to Difivac-l. Starting from the
determined position of the glycoprotein gE—gene and using the
cloned DNA fragments which flank the gE—gene, a gE deletion
fragment could be constructed. Using a standard technique
(F.L. Graham and A.J. van der Eb, 1973, Virology 52, 456-467),
this deletion fragment could be recombined in the genome of a
wild—type BHV—1 strain, resulting in a gE deletion mutant.
a) The construction of the gE deletion fragment
For the construction of the gE deletion fragment, a
lacks the
contains sufficient
fragment was aimed for which, on the one hand,
entire gE sequence and, on the other,
flanking sequence to allow recombination with the wild—type
At the 5' the 1.2 kb Pstl-AsuII
fragment which ends 18 nt before the start codon of the gB
For the 3'
genome. (upstream) side,
gene was chosen. (downstream) fragment the 1.2 kb
EcoNI-DraI fragment was chosen, which starts 2 nt before the
stop codon of the gE gene (Fig. 6).
For the construction of the gE deletion fragment, the
1.4 kb PstI-SmaI fragment coming from the 8.4 kb HindIII K
fragment of BHV-1 strain Lam, located at the 5‘ side of the gE
gene, was subcloned into the Smal and PstI site of plasmid
pUC18.
located on the 3'
This clone was called p515. The EcoNI—SmaI fragment
side of gE and coming from the 4.1 kb
HindIII—EcoRI clone was cloned into the unique AsuII site of
p515. Thus,
completed and the clone so constructed was called p519.
the construction of the gE deletion fragment was
Although in principle the entire PstI-SmaI insert of p519
this is not advisable.
100-150 base
into the repeat sequence which flanks the Us
could be used as gE deletion fragment,
The fact is that the PstI-SmaI extends approx.
(bp)
region.
pairs
This piece of 100-150 bp could recombine with the
repeat sequence on the other side of the Us area where the gE
gene is not located and could thus yield undesirable
recombination products. For that reason, the PstI-Dral
fragment was chosen for the recombination experiment, so that
bp of the repeat are removed.
b) Recombination of the gE deletion fragment with the genome
of wild—type BHV—1
In order to effect the recombination between the
constructed gE deletion fragment and the genome of wild—type
BHV—1, microgram amounts of the two DNA molecules are
cotransfected to Embryonal bovine trachea (Ebtr) cells
according to the standard method of F.L. Graham and A.J. van
der Eb (1973, Virology 52, 456-467).
mechanisms lead to the recombination of a small percentage of
the DNA molecules
Cellular recombination
(2—4%) which have been incorporated by the
cells. For the selection of the recombined gB deletion
mutants, the virus mixture that is formed after transfection
is disseminated on a fresh Ebtr cell culture. In most cases,
the separate virus populations which thereby develop (plaques)
originate from one virus. For the isolation of gE deletion
mutants of BHV-1 strain Lam, 230 of these plaques were
isolated and examined according to standard immunological
(Mabs)
These Mabs are
methods with BHV—1 specific monoclonal antibodies which
do not react with Difivac—l infected cells.
directed against the glycoprotein gE. Five of the 230 plaques
did not react with these Mabs. The DNA of these 5 plaques was
further investigated.
c) DNA analysis of the constructed gE deletion mutants of
BHV-l strain Lam
DNA preparations of 3 (1B7, 1B8 and 2Hl0)
mentioned 5 candidate gE deletion mutants were further
of the above
examined using the standard Southern blot analysis technique
(Sambrook et al.
preparations with PstI and DraI,
). Double digestions of these DNA
followed by gel
electrophoresis and Southern blot hybridization with the
.3 kb PstI—DraI deletion fragment as probe show that the gE
gene of the genome of virus populations 1B7 and 1B8 has been
removed exactly in the desired manner; see Figs 7A and 7B.
Population 2HlO has a deviant PstI—DraI fragment. Southern
blot hybridizations with a gE-specific probe show that no gE
sequences are located in any of the three DNA preparations
(results are not shown). BHV—l virus populations 1B7 and 1B8
BHV—l virus
population 1B7 has been tested for vaccine properties.
are intended recombinant gE deletion mutants.
d) Construction of thymidine kinase/gE double deletion
mutants
Because BHV—l recombinant deletion mutants with a
deletion in only one gene may not be of sufficiently reduced
virulence, deletions were also provided in the thymidine
kinase (TK) gene of the BHV—l strains Lam and Harberink. These
mutants were constructed in an analogous manner to that used
for the above-mentioned gE deletion mutants (results are not
shown). These TK deletion mutants have been used to construct
TK/gE double deletion mutants.
e) Construction of glycoprotein gI/ glycoprotein gE double
deletion mutants
Because cattle vaccinated with a single gE deletion
mutant may produce anti-gI antibodies that can interfere with
the detection of anti gI/gE antibodies (discussed below), we
also invented a vaccine with a gI/gE double deletion. Such a
gI/gE double deletion mutant can be constructed using the same
procedures used for the construction of the gE single deletion
mutant. Partial nucleotide sequence analysis of the upstream
end of the 1.8 kb Pstl fragment — that covers the 5‘ end of
the gE gene - revealed an open reading frame with significant
homology to g: homologs found in other herpesviruses. See
Figures 13 and 14. Using the 350 bp SmaI—PstI fragment that
encompasses the putative 5' end of the gI gene and the EcoNI—
SmaI fragment, located downstream of the gE gene, a gI/gE
deletion fragment can be constructed. This fragment can be
recombined with the wild type genome to yield a BHV-1 gI/gE
deletion mutant. See figure 16. The 80 — 90 amino acids that -
theoretically - may still be produced, will not be able to
elicit antibodies that can interfere with the detection of
anti-gI/gE antibodies. Further sequence analysis of the g1
gene will allow the construction of a gI deletion that covers
the complete gI coding region. This gI/gE double deletion
mutant has been named Difivac-IE.
f) Evaluation of safety and efficacy of the Lam gE' and the
Lam gE', TK' mutants
Vaccine properties of the Lam gE‘, and the Lam gE', TK'
BHV—l mutant strains were tested in seven—week-old, BHV—l
seronegative, specific pathogen free calves. Each mutant
strain was sprayed intranasally in 6 calves. Each calf was
given a total dose of 105 TCID5o in 2 ml culture medium, of
which 1 ml was sprayed in each nostril. Another 6 calves were
sprayed intranasally with virus-free culture medium, and
served as unvaccinated controls. Five weeks after vaccination
all calves, vaccinated and controls, were challenged intra-
nasally with 107 TCID5o of the highly virulent BHV—l strain
Iowa. After vaccination and after challenge, clinical signs,
rectal temperatures and body weight were monitored. Nasal
swabs were taken to determine the number of days of nasal
virus shedding.
After vaccination, behaviour, appetite, rectal
temperature and growth rates of the calves remained normal.
Serous nasal discharge and small lesions of the nasal mucosa
were observed in all vaccinated calves. Virus could be
isolated from the noses of the vaccinated calves for
approximately 7 days (Table 1).
After challenge, all unvaccinated control calves showed
apathy, loss of appetite, ocular and nasal discharge,
reddening of the gingiva of the lower jaw, severe lesions of
the nasal mucosae and growth was reduced. Calves vaccinated
with Lam gE‘, TK' all developed some nasal discharge and showed
some minor lesions of the nasal mucosae. Not all calves
vaccinated with Lam gE‘ did develop nasal discharge or lesions
of the nasal mucosae. Apathy, or other
loss of appetite,
clinical symptoms of disease were not observed with vaccinated
calves. Rectal temperature, growth and clinical score after
and 24.
shed virus from the nose 2 times longer than vaccinated calves
(Table l).
challenge are shown in Figs 22, Unvaccinated calves
The above results demonstrate that the Lam gB' and the
Lam gE‘, TK‘ BHV-1 mutant strains hardly induced any clinical
sign of disease in young calves. Both mutant strains prevented
sickness after challenge and reduced the period of nasal virus
shedding with 50%.
Lam gE' and Lam gE', TK‘ BHV-1 mutant strains are safe and
efficacious for use as a vaccine in cattle against BHV-1
infections.
) Prokaryotic expression of gE
For the prokaryotic expression of the BHV-1 glycoprotein
gE—gene,
(D.B.
so far use has been made of pGEX expression vectors
Smith and K.S. Johnson, Gene 67 (1988) 31—40). pGEX
vectors code for the carrier protein glutathione S-transferase
(GST) from Schistosoma japonicum which is under the influence
of the tac promoter which can be induced to expression by
Isopropylthiogalactoside (IPTG). An example of a GST-gE fusion
protein is the product of construct pGEX—2T600s3 (Fig. 8A). In
this construct, using standard molecular-biological techniques
(Sambrook et al. 1989), a 600 bp Smal fragment which codes for
an N-terminal region of 200 amino acids of the gE protein was
ligated behind the GST gene. This construct was designed in
triplicate, with each time a different reading frame of the
600 bp fragment being ligated to the GST. All three constructs
were introduced into Escherichia coli strain DH5a, induced
with IPTG and the proteins formed were transferred to
nitrocellulose after polyacrylamide gel electrophoresis by
means of Western blotting. Immunological detection with
anti—GST antibodies demonstrated that only the proper reading
frame (No. 3) which codes for the gE protein area leads to the
expression of a prominent fusion protein of the predicted size
of 27k (GST) + 20k (gE) = 47k. Three of the Mabs isolated by
us that do not react with Difivac-1 recognize the 47 kD GST—gE
fusion protein in a Western blot; see figure 8B.
) Eukaryotic expression of the glycoprotein gE-gene
For the eukaryotic expression of the glycoprotein
gE—gene, heretofore inter alia the vector pEVHis has been
chosen. The pEVHis vector has, as eukaryotic marker, the HisD
gene coding for the histidinol dehydrogenase [EC 1.1.1.23]
1988, Natl. Acad. Sci.
which causes cells to survive the toxic
(C. Hartmann and R. Mulligan,
85, 8047-8051)
concentration of 2.5 mM histidinol. The vector moreover
Proc. USA
comprises the promoter region of the immediate early gene of
the human cytomegalovirus (HCMV), with unique restriction
enzyme sites located behind it. For the construction of a
pEVHis/gE expression vector, use was made of a fragment
comprising the entire coding region of the glycoprotein
It starts on the Alul site 55 bp before the
postulated open reading frame of gE and ends 133 bp behind it.
gE-gene.
This region was cloned behind the HCMV promoter of the pEVHis
vector, whereby the construct pEVHis/gE was formed (Fig. 9).
The pEVHis/gE was amplified in E.coli DH5a cells and purified
by means of a cesium chloride gradient (Sambrook et al.,
1989). This purified DNA was transfected to Balb/C-3T3 cells
according to the method of Graham and Van der Eb. Transformed
cells were selected with histidinol, whereafter twenty
histidinol resistant colonies could be isolated. These
colonies were examined with Mab 81 by means of an Immuno
Peroxidase Monolayer Assay (IPMA). Four colonies proved to
express the gE protein. Of these four colonies, 3T3 gE clone 9
was used to isolate a subclone having a high gE expression.
The clone isolated by this method (called 3T3gE 9.5) was used
for characterizing candidate anti—gE monoclonal antibodies.
) Eukaryotic expression of both the BHV—l glycoprotein gE
and the BHV—1 glycoprotein gI in the same cell
To express the BHV—1 glycoprotein gI in the same cell as
the BHV—1 glycoprotein gE we first determined the putative
position of the BHV—1 gl gene. Because the herpes simplex
virus glycoprotein gI gene is located just upstream of the
glycoprotein gE gene, it was inferred that the BHV-1 gI gene
would be located on a corresponding position. To test this,
the sequence has been determined of a region of 283
nucleotides, located about 1 kb upstream of the start of the
BHV-1 gE gene. Conceptual translation of this region showed
that the second reading frame codes for a 94 amino acids
sequence that is homologous to the herpes simplex virus
glycoprotein gI (figures 13 and 14). Because the homologous
segment is about 80 amino acids from the start codon the
putative start of the open reading frame of the BHV-1 gI gene
is estimated about 250 nt upstream from the sequenced region.
From this it was inferred that the 1.7 kb SmaI fragment that
starts 400 nt upstream from the sequenced region and ends
within the gE gene should contain the complete coding region
of the BHV-1 gI gene. This 1.7 kb Smal fragment has been
cloned into the eukaryotic vector MSV-neg (See figure 15).
This vector contains the strong Murine Sarcoma Virus promoter
and the selector gene neg that codes for the resistance
against the antibiotic G—4l8 sulphate Geneticin. The resulting
construct MSVneoGI has been amplified in E. coli DH5a cells
and has been transfected into 3T3gE 9.5 cells using the method
of Graham and Van der Eb. The transfected cells were selected
with 400 ug Geneticin/ml culture medium and the resistant
colonies have been isolated and tested with candidate anti—gE
Mabs that failed to react with 3T3gE 9.5 cells. From this we
selected the 3T3gE/gI R20 clone that reacted with e.g. Mab 66
as well as wild type BHV-1 does.
) Characterization of candidate anti—gE Mabs
Mabs were produced against wild-type BHV-1 and selected
for their inability to react with Difivac—l infected embryonic
bovine trachea (Ebtr) cells. These Mabs are examined for their
reactivity with
a) the Lam gE' deletion mutant;
b) the above described prokaryotic expression product in a
Western blot;
c) the above described gE—expressing Balb/c—3T3 cells;
d) cells mentioned under c) and
and infected with Difivac—l,
e) Balb/c-3T3 cells expressing the gE/gI complex.
For reactivity testing under a, c, d and e an immuno-
(IPMA)
Table 2 show that we have produced Mabs that are directed
(nrs. 2, 3, 4, 52, 66, 68, 72 and 81) and Mabs
(nrs. 1, 51, 53, 67, 75 and 78) that may be directed against
peroxidase monolayer assay was used. The results in
against gE
conformational antigenic domains on the gE/gI complex. A
competition IPMA to map the antigenic domains recognized by
the various Mabs indicated that at least 4 antigenic domains
are present on glycoprotein gE and that one domain probably is
formed by the gE/gI complex (Table 2).
Detection of anti-gE antibodies in cattle infected by BHV—1
To examine whether in serum of infected cattle antibodies
against gE are present an indirect blocking IPMA was performed
with the 16 candidate gE—Mabs and the following 8 selected
sera:
- 2 sera of bovines vaccinated with Difivac-l and challenged
with the virulent Iowa strain, that were collected 14 days
after challenge;
- 2 sera of bovines experimentally infected with BHV-1 subtype
virus, that were collected 20 months after infection. One of
the bovines was infected by contact exposure;
— 2 sera of bovines experimentally infected with BHV-1 subtype
2b virus, that were collected 20 months after infection. One
of the bovines was infected by Contact exposure;
- a serum of a specific pathogen free calf vaccinated with a
ts mutant vaccine and challenged 3 weeks later with BHV-1
subtype 2b virus, that was collected 7 weeks after challenge;
— a serum of a gnotobiotic calf vaccinated with a ts mutant
vaccine and challenged 3 weeks later with BHV—l subtype 2b
virus, that was collected 7 weeks after challenge.
Table 2 shows that all these sera contained antibodies
against the antigenic domains III and IV on gE, and against
antigenic domain I that is probably located on the gE/gI
complex. We may conclude that gE appears to be a suitable
serological marker to distinguish between BHVinfected and
vaccinated cattle.
) Detection of BHV-1 nucleic acids by means of the PCR
procedure using BHV-1 gE-specific primers
Starting from the determined nucleotide sequence of the
BHV-1 gE gene, a primer pair suitable for the PCR was
selected, using the primer selection program by Lowe et al.
J. Sharefkin, S. Qi Yang and C.W. Dieffenbach, 1990,
Nucleic Acids Res. 18, 1757-1761). These primers were called
(T. Lowe,
P3 and P4 and are shown in Fig. 10. The primers are located
159 nt apart and lead to the amplification of a fragment of
200 nt. Using primers P3 and P4 and isolated BHV-1 DNA, the
conditions for the PCR procedure were optimized. This involved
in particular the variation of the MgCl2 concentration, the
glycerol concentration and the cycling conditions. The optimum
buffer found for the use of P3 and P4 for the amplification of
BHV-1 DNA is 10 mM Tris pH 8.0, 50 mM KCl, 0.01% gelatin, 2.6
mM MgCl2 and 20% glycerol. The optimum cyclic conditions found
(Perkin Elber Cetus DNA Thermal Cycler)
98°C, 55°C and 45 sec. 72°C and for cycli 6-35:
96°C, 55°C and 45 sec. 72°C. After the PCR
amplification, the 200 nt DNA fragment obtained was
are for cycli 1-5:
1 min. 30 sec.
sec. 30 sec.
electrophoresed on a 2% agarose gel, blotted on nitrocellulose
and subsequently subjected to Southern blot analysis. The 33P
dCTP labeled probe used for the Southern blot analysis is the
137 bp Taql fragment which is located between the primer
binding sites (Fig. 10). After autoradiography of the
hybridized filters, a 200 bp band can be observed. Via this
1.5 x
“15 ug DNA) still leads to a properly detectable signal
route, amplification of only 10 BHV-1 genomes (approx.
(result not shown). In a comparable manner, a PCR procedure
was developed using primers which are based on the coding
sequence of the BHV-1 glycoprotein gIII (D.R. Fitzpatrick,
L.A. Babiuk and T. Zamb, 1989, Virology 173, 46-57).
a distinction to be made between wild—type BHV-l DNA and a gE
To enable
deletion mutant vaccine, DNA samples were subjected both to
the gE—specific PCR and to gIII—specific PCR analysis. In such
a test, a Difivac—l DNA preparation was found to be gIII
positive and gE negative.
Because the detection of BHV-l DNA in bovine semen will be an
important use of the BHV-l specific PCR procedure, it was
attempted to perform the gE—specific PCR on bovine semen
infected with BHV-l. However, unknown components in the semen
have a strongly inhibitory effect on the polymerase chain
reaction. Therefore, a protocol was developed to isolate the
BHV-l DNA from bovine semen. To isolate the DNA from bovine
semen, 30 ul of semen is incubated with 1 mg/ml proteinase K
(pK) in a total volume of 300 ul O.l5M NaCl, 0.5% Na-Sarkosyl
and 40 mM DTT, at 60°C. After 1 hour the sample is allowed to
cool down to room temperature and 300 ul 6M NaI is added and
incubated for 5 min. From this mixture DNA is isolated with a
standard chloroform/isoamylethanol extraction and precipitated
with 1 volume isopropanol. The precipitate is washed with
.5 M NH4Ac/70% ethanol and resuspended in 10 mM Tris pH7.4,
1mM EDTA, 0.5% Tween 80 and 0.1 mg/ml pK for a second
incubation for 1 hour at 60°C. This DNA preparation can be
directly submitted to the Polymerase Chain Reaction.
DESCRIPTION OF THE DRAWINGS
Figure 1
Southern blot analysis of BHV-l strains Difivac—1 and Iowa
A. Drawing of an autoradiogram of a Southern blot of
Difivac-1 and Iowa genomic DNA. In lanes 1 and 3, Difivac-1
DNA was applied after restriction enzyme digestion with
HindIII and PstI, respectively. In lanes 2 and 4, Iowa DNA was
applied after restriction enzyme digestion with HindIII and
PstI, respectively. The size of the fragments is indicated in
kilobase (kb).
Viral DNA was isolated by centrifuging the culture medium
(70 ml/roller bottle of ca. 450 cm?) with virus infected Ebtr
cells for 2 h through a 25% (w/w) in 10 mM
Tris pH 7.4, 150 mM NaCl and 1 mM EDTA at 20 krpm in the SW27
rotor of the Beckman L5—65 ultracentrifuge. From the virus
sucrose cushion,
pellet so obtained, DNA was isolated according to standard
methods (J. Sambrook, E.F. Fritsch and T. Maniatis, 1989,
Molecular cloning: a laboratory manual, 2nd ed. Cold Spring
Harbor Laboratory Press, New York). On this DNA, restriction
enzyme digestions were performed with enzymes from Boehringer
Mannheim in the SuRE/cut buffers supplied by the manufacturer.
After separation on a 0.7% agarose gel for horizontal
electrophoresis and blotting on a nitrocellulose filter
(Schleicher & Schuell,
6 h at 42°C in 50% formamide,
.015 M Na—citrate, pH 7.4),
Inc.) the filter was prehybridized for
3x SSC (lx SSC = 0.15M NaCl and
50 ul denatured salmon sperm DNA
(Sigma)/ml and 0.02% bovine serum albumin, 0.02% polyvinyl
pyrrolidone and 0.02% ficoll and 0.l% Na-dodecylsulphate
(SDS). Then, hybridization was performed by adding to the same
solution the 32P dCTP (Amersham) labeled HindIII K fragment
(The choice of the HindIII K fragment is based on: Cloning and
cleavage site mapping of DNA from bovine herpesvirus 1 (Cooper
strain), John F. Mayfield, Peter J. Good, Holly J. Vanoort,
Alphonso R. Campbell and David A. Reed, Journal of Virology
(l983) 259-264). After 12-14 h hybridization, the filter was
washed for 2 h in 0.1% SDS and 0.1 X SSC at 60°C. The HindIII
K fragment was cloned into the pUC18 vector according to
standard cloning procedures (J. Sambrook, E.F. Fritsch and
T. Maniatis, 1989, Molecular cloning: a laboratory manual, 2nd
ed. Cold Spring Harbor Laboratory Press, New York). After
HindIII digestion of the pUC/8.4 HindIIIK clone the pUC18
vector was separated from the 8.4 kb HindIII K fragment again
by electrophoresis on a 0.7% Low Melting Point Agarose (BRL,
Life Technologies, Inc.) gel, and isolated from the agarose by
standard phenol extraction and ethanol precipitation. The
isolated HindIII K fragment was labeled with the Random Primed
DNA labeling Kit lOO4.76O from Boehringer Mannheim.
Autoradiography of the hybridized filters was carried out
through 36 h exposition of a Kodak XAR film at —70°C, using a
reflecting screen.
B. Physical maps of the 8.4 kb HindIII K fragment of Iowa
and of the 7.4 kb HindIII fragment of Difivac-1. In view of
the comigration of the 6 kb PstI fragments and the absence of
the 1.8 kb Pstl fragment in Difivac—l, the deletion is
postulated in the hatched area.
Figure 2
Subcloning of wild-type BHV-1 fragments around the region
lacking in Difivac-l
In A the components of the BHV—l genome are shown: The
(UL) (Us)
(Ir and Tr). This map is based on the published
analysis of the Cooper strain (John F. Mayfield, Peter J.
Unique Long region; the Unique Short region and the
two repeats
Good, Holly J. Vanoort, Alphonso R. Campbell and David A.
(1983) 259-264).
In B the fragments are shown from the Us region which
Reed, Journal of Virology
have been cloned into prokaryotic vectors: A 15.2 kb EcoRI
fragment in pACYC, an 8.4 kb HindIII fragment in pUCl8 and a
2.7 kb and a 4.1 kb EcoRI-HindIII fragment in pBR322. The
isolation of the viral DNA fragments was carried out according
to the procedures which are mentioned in the legends of Fig.
1A. The cloning of these fragments into the various vectors
(J. Sambrook,
1989, Molecular cloning: a
was carried out according to standard procedures
E.F. Fritsch and T. Maniatis,
laboratory manual,
New York).
nd ed. Cold Spring Harbor Laboratory
Press,
In C a physical map is shown of the region where the
postulated deletion in Difivac-l is localized.
which
were used for further analysis. The two Pstl fragments were
In D some subclones of this region are indicated,
cloned into pKUNl9 and the remaining fragments into pUCl8.
Figure 3
A: Nucleotide sequence of 2027 nucleotides from the Us
region of BHV—1 strain Lam around the postulated location
which has been deleted in Difivac-1, as indicated in Fig. 2C
[from the Alul recognition site on the extreme left to the
HincII recognition site on the extreme right]. The nucleotide
sequence in the inserts of the subclones shown in Fig. 2D was
determined by analyzing on the two strands using the dideoxy
sequence method of Sanger et al. (F. Sanger, S. Nicklen and
1977, Proc.Natl. Acad. Sci. USA 74, 5463-5467).
To that end, the T7 sequence kit of Pharmacia was used
A.R. Coulson,
according to the procedure specified by the manufacturer. For
[358] dATP (Amersham) was used. The
sequence analysis of the GC rich regions with compression
the radioactive labeling,
artefacts was repeated with the 7—deaza—dGTP variant of the
Pharmacia kit. Indicated beneath the nucleotide sequence is,
in the three—letter code, the amino acid (aa) sequence of the
open reading frame of 575 aa residues, which was found after
conceptual translation of the nucleotide sequence. This
translation is based on the universal code and was determined
using the PC/gene computer program (PC/gene version 1.03,
November 1987). This open reading frame of 575 aa starts with
the methionine at nt 168 and ends with the stop codon at
nucleotide 1893.
Structural analysis of the open reading frame of 575 aa
residues was also performed with the PC/gene computer program.
The first 26 aa form a eukaryotic export signal indicated in
the
cleavage of this signal sequence is predicted between aa 26
the figure by "signal peptide". with a score of 6.2,
and aa 27. The sequence of 575 aa has 3 possible N-bound
(NXT/S)
amino acid residues. According to the Rao and Argos method
glycosylation sites indicated by a line under the
there is a transmembrane region between aa 423 and aa 450
indicated in the figure by "transmembrane helix". Recognition
sequences (sites)
for the restriction enzymes AsuII, Smal,
HindIII and EcoNI are underlined. The calculated molecular
weight of this polypeptide is 61212.
B: Schematic representation of the structural charac-
teristics of the above mentioned 575 aa open reading frame.
Figure 4
Amino acid comparison of the amino acid sequence of the BHV—1
gE gene with the amino acid sequence of the herpes simplex
(HSV)
virus gE gene and other gE homologous genes
(PRV)
[pseudo-
(VZV) GPI]
The sequences used for this comparison come from the
rabies virus g1 and varicella-zoster
following publications; HSV: Sequence determination and
genetic content of the short unique region in the genome of
herpes simplex virus type 1. D.J. McGeoch, A. Dolan, S. Donald
(1985) Journal Mol. Biol. 181, 1-13. VZV: DNA
sequence of the Us component of the varicella-zoster virus
(1983), EMBO Journal 2, 2203-2209. PRV:
Use of lgtll to isolate genes for two pseudorabies Virus
and F.J. Rixon
genome. A.J. Davidson
glycoproteins with homology to herpes simplex virus and
varicella-zoster virus glycoproteins. E.A. Petrovskis, J.G.
(1986) 185-193].
These sequences were compared using the sequence analysis
1988, Nucl. Acids Res. 16,
Timmins and L.E. Post Journal of Virology 60,
program Multalin
10881-10890).
In A a diagram is shown in which all four amino acid
(F. Corpet,
sequences are shown schematically. Here, the predicted
transmembrane parts (TM) are shown below each other. In
addition to the predicted export signal sequences (SP) and the
(I);
in which the relative position of the cysteine
(C C C).
In B the results are shown of the Multalin comparison of
possible N—bound glycosylation sites two conserved areas
are shown,
residues is often unchanged
the centrally located cysteine rich region of the four gE
versions. Asterisks indicate identical amino acids and colons
analogous amino acids.
Figure 5
Drawing of photographs obtained in a Southern blot analysis of
Difivac-1 and Iowa
Panel A: Genomic DNA of Difivac—1 and Iowa restriction
(1,2), BcoRI (3,4) and HindIII
separated on a 0.7% agarose gel, blotted on nitro-
enzyme digestions with Bstl
(5,6)
cellulose and hybridized with 32? labeled HindIII K fragment of
BHV-1 strain Lam according to the procedures specified in the
legends of Fig. 1A.
Panel B: Nitrocellulose blot of the same gel as in A
hybridized with the BHV-1 gE—specific probe p318. This probe
comprises the entire AluI—HincII region indicated in Fig. 2C.
Figure 6
Construction of gE deletion fragment BHV-1
In A the position of the gE gene and the clones used is
shown. The components of the BHV-1 genome are:
(UL) (Us)
(IR and TR). To obtain the region located on the 5'
The Unique Long
region; the Unique Short region and the two repeats
side of
the gE gene, the 1.4 kb Pstl-Smal fragment from the 8.4 kb
HindIII K fragment of BHV-1 strain Lam was subcloned into the
Smal and Pstl site of plasmid pUC18. This clone was called
p515 and is shown in B. The EcoNI-SmaI fragment located on the
3' side of gE, coming from the 4.1 kb HindIII-EcoRI clone was
cloned into the unique AsuII site of p515. To enable the
ligation of the EcoNI rest to the AsuII rest, clone p515 was
digested with AsuII, then treated with Klenow enzyme
(Boehringer Mannheim) and dCTP to provide one cytosine residue
(Sambrook et
). This additional cytosine is indicated by an
in the AsuII rest according to standard methods
al.,
asterisk in D. Then, p515 was also digested with the SmaI
enzyme, whereafter the EcoNI fragment could be ligated into
this vector. The clone thus constructed was called p519.
Figure 7
A. Drawing of a photograph obtained in Southern blot
B8 and 2H10. DNA
blotting and
hybridization were performed according to the procedures
lA. After Pstl-DraI double
1B8 and 2HlO, the
fragments were separated on a 0.7% agarose gel and
analysis of DNA preparations of lB7,
isolation, restriction enzyme digestions,
described in the legends of Fig.
digestion of the DNA preparations 1B7,
subsequently blotted on a nitrocellulose filter. This filter
was hybridized with the 32}? dCTP labeled 2.3 kb PstI—DraI
deletion fragment as probe.
1B7,
In lanes 1 through 3, the samples
lB8 and 2H1O were separated, respectively. In lane 4,
wild-type BHV—l DNA of the Lam strain was applied and in lane
the 2.3 kb deletion fragment.
B. Physical map of the 15.2 kb EcoRI fragment of BHV—1
strain Lam. The map shows the position of the PstI, DraI and
HindIII recognition sites and the position of the
hybridization probe mentioned in 7A.
Figure 8
Prokaryotic expression of BHV—l gE
For the prokaryotic expression of BHV-1 gE, the 600 bp
SmaI fragment of the gE gene was fused in three reading frames
to the coding region of the glutathione-S—transferase gene
(D.B. Smith
Recombinant molecules
from Schistosoma japonicum in the vector pGEX—2T
Gene 67 (1988) 31-40).
(syn) orientation of the Smal fragment were
identified by means of restriction enzyme analysis using
standard methods. E. coli DH5a clones with this fusion
construct were called pGEX-2T600sl, pGEX—2T600s2 and
pGEX—2T600s3.
and K.S..Johnson,
with the proper
A. Diagram of one of the pGEX-2T600s constructs. Located
on the NH; side of the region which codes for GST-gE fusion
(IPTG)
product is the Isopropylthiogalactoside inducible tac
promoter region.
B. Drawing of photographs obtained in Western blot
analysis of total protein preparations of DH5a cells
transformed with pGEX—2T600s. Overnight cultures of DH5a cells
transfected with the constructs pGEX—2T600sl, pGEX—2T600s2 and
pGEX—2T600s3 were continued 1/10 in Luria—Bertani (LB) medium
with 50ug/ml ampicillin and after 1 h growth induced with IPTG
for S h. These induced cultures were centrifuged for 5 min at
6,000 x g and incorporated in l x layermix (2% SDS, 10%
Glycerol, 5% mercaptoethanol and 0.01% bromophenol blue)
[l.5 ml culture is incorporated in 500 ul layermix] and heated
at 95°C for 5 min. Then 50 ul per lane was separated on a
vertical 12.5% polyacrylamide gel according to standard
procedures and subsequently Semi—dry blotted to a
nitrocellulose filter using the LKB—multiphor II Nova Blot
system under the conditions specified by the manufacturer.
In lanes M, prestained marker protein was applied (BRL
236k, 112k, 71k, 44k, 28k, 18k and
and 3 the total protein preparations of
Life Technologies, Inc.
15k)
DH5a cells transfected with the three respective frames:
pGEX—2T600sl, pGEX-2T600s2 and pGEX-2T600s3.
In panel A, the result can be seen of the western blot
and in lanes 1,
analysis with anti-GST serum. To that end, the filter was
incubated according to standard procedures (5. Harlow and D.
1988, Antibodies: a laboratory manual, Cold Spring
Harbor Laboratory, New York) (PBS + 2% milk
powder and 0.05% Tween 20) and subsequently with polyclonal
Lane,
in blocking buffer
anti-GST rabbit serum. Then the filter was washed and
incubated with horse radish peroxidase (HRPO) conjugated
goat—anti~rabbit immunoglobulin serum. Then the bound goat
antibodies were immunochemically detected with chromogen
chloronaphthol and H202). The GST fusion
product which is indicated by an arrow has the predicted size
(diaminobenzidine,
of approx. 47 k only in frame 3.
In panel B, the result can be seen of the western blot
analysis with monoclonal antibody Mab 4, which recognizes the
gE protein. To that end, a duplo filter as in panel A was
blocked, and incubated with HRPO
the bound rabbit
antibodies were immunochemically detected with chromogen. The
incubated with Mab, washed,
conjugated rabbit—anti-mouse serum. Then,
band which is visible in lane 3
(frame 3) is 47k in size and
is indicated by an arrow.
Figure 9
Construction of the pEVHisgE plasmid for the eukaryotic
expression of the BHV—l gE gene
For the eukaryotic expression of the gE gene, the entire
gE coding region was cloned in the proper orientation behind
the HCMV promoter region of the expression vector pEVHis using
(Sambrook et al. 1989). To that end, the
bp Alul fragment which starts 55 bp before the open
standard procedures
reading frame of the gE was cloned into pUC18 and called p201.
Then, after HincII digestion of p201, the 1740 bp HincII
fragment, which comprises the greater part of the gE gene, was
cloned into p201. This resulted in the plasmid p318 which in
the polylinker of pUC18 comprises the entire gE coding area
from the Alul site 55 bp before the start codon of gE to the
HincII site 133 bp behind the stop codon of gE. Using the
restriction enzyme sites in the polylinker of the vector, this
fragment was cut from p 318 with the enzymes BamHI and Sphl.
First, p318 was digested with Sphl and then the SphI site was
After the
digestion with BamHI, the 1.9 kb insert was separated from the
filled in using Klenow polymerase and dNTP's.
pUC18 vector in Low Melting Point Agarose and ligated in the
pEVHis vector which had been digested with BamHI and EcoRV to
that end. The plasmid so formed was called pEVHis/gE.
Figure 10
Position of the gE-specific primers and probe for the PCR
procedure for detecting BHV—l DNA
Shown in the figure is the nucleic acid sequence of the
BHV-1 glycoprotein gE gene from nucleotide 1272 to 2027 [the
sequence has been taken over from Fig. 3]. The primers used
for the gE—specific PCR procedure were called P3 and P4. The
primer binding sites for P3 and P4 are underlined. The
nucleotide sequence of P3 is 5'-ACG—TGG—TGG—TGC-CAG—TTA—GC-3'
(SEQ ID NO:2). The nucleotide sequence of P4 is (complementary
‘—ACC—AAA-
(SEQ ID NO:3). The probe which was
used for the Southern blot hybridization for the detection of
the PCR amplified DNA,
to the primer binding sequence specified above)
CTT-TGA-ACC-CAG-AGC-G-3‘
is the 137 bp TaqI fragment located
between the primer binding sites, the ends of this fragment
being indicated. For comparison with Fig. 3, the HindIII and
the EcoNI sites are also indicated.
Figure 11
Mapping of the gE deletion of Difivac—1
A shows the physical map of the 15.5 kb EcoRI fragment of
the wild type BHV-1 strain Lam. B shows the physical map of
the 14.5 kb EcoRI fragment of Difivac—l. Both EcoRI fragments
cover the complete Unique short regions of the genomes of the
respective viruses. The position of the gE gene and the
putative position of the gI gene have been indicated by open
boxes. Maps A and B are positioned in such a way, that the
6 kb PstI fragments within each map are aligned. In both maps
the internal repeat and the terminal repeat sequences have
been indicated by hatched boxes. The arrows beneath the
repeats indicate the orientation of these sequences.
In A the part of the Us region that is missing in the
Difivac—l strain has been indicated.
C shows the position of the cloned Difivac-1 fragments
used to map the gE deletion and to obtain the physical map
shown in B. The arrows beneath the inserts of clones p728,
p737 and p754 indicate the regions that have been sequenced to
determine the recombination point.
Abbreviations:
A = AluI, E = EcoRI, P = Pstl, H = HindIII, r = recombination
point, IR = internal repeat, TR = terminal repeat.
Figure 12
Determination of the exact recombination point in the Us
region of Difivac-l
To determine the exact borders of the gE deletion found
in the Difivac—l strain,
p728
have
have been described in the legends of figure 3.
clone p754 and the ends of clones
and p737 have been sequenced. The inserts of these clones
been indicated in figure 11. The sequence procedures used
In A the sequence of most of the Alul — PstI fragment has
been
shown. This sequence starts in the promoter region of the
gE gene. A putative TATA box has been underlined. At point r
(= recombination point) this promoter region is
sequence also found at the opposite site of the
named: inverted repeat. The exact recombination
determined by comparing the repeat found at the
region with the copy of the repeat found at the
of the Us region. The point were these sequences
been indicated in B with
(under I) 'r'.
fused to a
Us region,
point has been
gE promoter
opposite site
diverge has
A similar comparison
has been made with the gE promoter sequence found in Difivac—l
and the gE promoter found in wild type strain Lam. The point
were these sequences diverge has been shown in B (under II)
and also indicated with 'r'.
are the same.
Figure 13
Partial sequence analysis of the BHV-l gI gene
Using the 1.8 kb PstI clone of BHV1 strain
reaches into both the BHV—1 gI and gE gene (See
The recombination points found
Lam, that
figure 11),
the sequence of 284 nucleotides within the coding region of
BHV—l gI was determined. The sequence procedures used have
been described in the legends of figure 3.
The sequence has
been translated based on the universal code by the PC/gene
computer program version 1.03 1987).
sequence encoded by the second reading frame is
(Nov.
one letter code beneath the nucleotide sequence.
The amino acid
given in the
This amino
acid sequence is homologous to the coding region of other
herpes virus gI homologs (See figure 14).
Figure 14
Amino acid comparison of the partial amino acid sequence of
the putative BHV—1 gI gene with the corresponding parts of the
(HSVl) gI gene, the
gp63 gene and the varicella—zoster
coding regions of the herpes simplex virus
pseudorabies virus (PRV)
virus (VZV) gpIV gene.
the HSV1
sequence starts at aa 80 and the VZV sequence starts at aa 76
The PRV sequence starts at amino acid 82,
of their respective coding regions. The sequences used were
published in the papers mentioned in the legends of figure 4.
The comparison was performed using the Multalin computer
program. Asterisks indicate identical amino acids and colons
indicate analogous amino acids.
Figure 15
Construction of the MSVneoGI plasmid for the eukaryotic
expression of the BHV-l gI gene
Based on the amino acid comparison of the partial
sequence of the BHV-1 gI gene, the putative position of the
BHV-1 gI gene has been estimated. Based on this estimation it
was inferred that the 1.7 kb Smal fragment should contain the
complete coding region of the BHV-1 gE gene. The position of
this 1.7 kb Smal fragment has been indicated in A. To the
blunt ends of this 1.7 kb Smal fragment, BamHI linkers have
been ligated, using standard procedures. The resulting product
was digested with BamHI and ligated into the eukaryotic
expression vector MSV-neo. The MSV-neo vector has a unique
BamHI site behind the MSV-LTR, which has a strong promoter
activity. This vector has been described in Rijsewijk et al.,
1987 EMBO J. 6, 127-131.
Figure 16
Construction of a BHV—l gI/gE double deletion fragment
The position of the glycoprotein gE gene and the putative
position of the glycoprotein gI gene in the Us region of BHV-1
are depicted in diagram A. The hatched blocks indicate the
repeats that border the U3 region. B shows the physical map of
some essential restriction enzyme sites with respect to the
position of both genes. To construct the gI/gE deletion
fragment clone p1.7—SmaI/o containing the 1.7 kb SmaI fragment
that embraces the g1 gene will be digested with PstI. The PstI
site of the remaining 350 bp SmaI—PstI insert will be made
blunt ended using standard molecular biological procedures.
The EcoNI-SmaI fragment (see Figure 6B), isolated from the
4.1 kb HindIII-EcoRI fragment described in Figure 6A, will
also be made blunt ended and ligated to the modified PstI
site. This is diagrammed in C and D. From the resulting clone
pAIE the 1.4 kb SmaI—DraI fragment can be isolated to
recombine with wild type BHV-l DNA.
Abbreviations:
= ECORI, H = HindIII, S = SmaI, P = PstI, ENI = ECONI,
= Dral, kb = kilobase and Us = unique short.
Figure 17
Mean nasal virus shedding from calves after vaccination
- = vaccinated with Difivac-1, 0 = unvaccinated control.
Figure 18
Mean daily clinical score of calves after challenge with a
virulent BHV—l strain, key as in fig. 17.
Figure 19
Mean rectal temperature of calves challenge with a virulent
BHV—l strain, key as in fig. 17.
Figure 20
Mean growth of calves after challenge with a virulent BHV—l
strain, key as in fig. 17.
Figure 21
Mean nasal virus shedding from calves after challenge with a
virulent BHV-l strain, key as in fig. 17.
Figure 22
Mean rectal temperature of calves after challenge with a
virulent BHV—l strain
° = vaccinated with Lam gE‘, 0 = vaccinated with Lam gE‘/TK',
x = unvaccinated control.
Figure 23
Mean growth of calves after challenge with a virulent BHV-l
strain, key as in fig. 22.
Figure 24
Mean daily clinical score of calves after challenge with a
virulent BHV-1 strain, key as in fig. 22.
IAELE_l
Nasal virus shedding of calves after vaccination with Lam gE‘
or Lam gE‘/TK‘ and after challenge with a virulent BHV—1 strain
of these vaccinated and control calves
Average number of davs of nasal virus shedding
Group After vaccination After challenge
Control 0 10.33 i 1.51
Lam gE' 7.00 i 0.89 4.83 i 1.17
Lam gE'/TK‘ 7.17 i 1.33 5.17 i 0.98
IAELE_2
Characterization of gE-Mabs
Mab REACTIVITY OF CANDIDATE gE-Mabs WITH
Difi— Lam Prok. 3T3 gE 3T3 gE 3T3 Ag Ab
vac-1 gE' Difi— gE/gI group cattle
3T3/ vac-1
EBTR
- - nd - + ? I +
2 — — — + + + II — + + + + ? -
4 — - + + + + ? -
— — nd - — ? V? i
— — nd — + + III +
— - + + + + ? -
— — nd - + + III +
- - nd - - + III +
— - nd + + + III +
- — nd — + + III +
- — — + + + IV +
— — - + + + V i
— — nd — + ? I +
- - nd — + ? nd —
- — — + + + II? -
+ : All 8 tested sera score a blocking percentage of > 50% in
an indirect blocking IPMA.
Sera score a blocking percentage of i 50%.
- : Sera score a blocking percentage of < 50%.
SEQUENCE LISTING
SEQ ID NO:l
LENGTH: 2027 nucleotides, 575 amino acids
TYPE: nucleotide and amino acid
STRANDEDNESS: single
AGGGCGGAGC GTTGAGCGGC CCGACCGCCG CCGGGTTGTT AAATGGGTCT CGCGCGGCTC
---—> deleted in Difivacl
GTGGTTCCAC ACCGCCGGAG AACCAGCGCG AGCTTCGCTG CGTGTGTCCC GCGAGCTGCG
ATG CAA CCC ACC GCG CCG CCC CGG CGG CGG TTG CTG CCG CTG CTG CTG
Met Gln Pro Thr Ala Pro Pro Arg Arg Arg Leu Leu Pro Leu Leu Leu
1 5 10 15
SIGNAL PEPTIDE ====-==-====-===
CCG CAG TTA TTG CTT TTC GGG CTG ATG GCC GAG GCC AAG CCC GCG ACC
Pro Gln Leu Leu Leu Phe Gly Leu Met Ala Glu Ala Lys Pro Ala Thr
25 30
SmaI
GAA ACC_CCG_GGC TCG GCT TCG GTC GAC ACG GTC TTC ACG GCG CGC GCT
Glu Thr Pro Gly Ser Ala Ser Val Asp Thr Val Phe Thr Ala Arg Ala
40 45
GGC GCG CCC GTC TTT CTC CCA GGG CCC GCG GCG CGC CCG GAC GTG CGC
Gly Ala Pro Val Phe Leu Pro Gly Pro Ala Ala Arg Pro Asp Val Arg
50 55 60
GCC GTT CGC GGC TGG AGC GTC CTC GCG GGC GCC TGC TCG CCG CCC GTG
Ala Val Arg Gly Trp Ser Val Leu Ala Gly Ala Cys Ser Pro Pro Val
65 70 75 80
CCG GAG CCC GTC TGC CTC GAC GAC CGC GAG TGC TTC ACC GAC GTG GCC
Pro Glu Pro Val Cys Leu Asp Asp Arg Glu Cys Phe Thr Asp Val Ala
85 90 95
CTG GAC GCG GCC TGC CTG CGA ACC GCC CGC GTG GCC CCG CTG GCC ATC
Leu Asp Ala Ala Cys Leu Arg Thr Ala Arg Val Ala Pro Leu Ala Ile
100 105 110
GCG GAG CTC GCC GAG CGG CCC GAC TCA ACG GGC GAC AAA GAG TTT GTT
Ala Glu Leu Ala Glu Arg Pro Asp Ser Thr Gly Asp Lys Glu Phe Val
115 120 1
120
CTC
Leu
GTG
Val
145
TAC
Tyr
CTG
Leu
GAG
Glu
CGC
Arg
GTG
Val
225
CTA
Leu
GCC
Ala
ATC
Ile
CTG
Leu
GAG
Glu
305
GCC
Ala
130
CTG
Leu
GAC
Asp
ACG
Thr
GAG
Glu
ACG
Thr
210
CTG
Leu
TCG
Ser
AGC
Ser
CGC
Arg
CAC
His
290
ACC
Thr
GAC
Asp
ATC
Ile
CGG
Arg
CTG
Leu
AGG
Arg
195
ACG
Thr
CCG
Pro
GTG
Val
ATC
Ile
ATA
Ile
275
CCC
Pro
GTG
Val
CCG
Pro
GCG
Ala
CTC
Leu
CAG
Gln
180
GAA
Glu
ACA
Thr
TAC
Tyr
CGT
Arg
GAC
Asp
260
TAC
Tyr
GCC
Ala
TAC
Tyr
CAC
His
GCC
Ala
ATC
Ile
165
GTC
Val
CCA
Pro
CGC
Arg
CAC
His
CTG
Leu
245
TGG
Trp
GAG
Glu
GAC
Asp
AGC
Ser
GTC
Val
GCA
Ala
150
GGC
Gly
GCG
Ala
GCG
Ala
GCG
Ala
Ser
230
CAG
Gln
TAC
Tyr
ACG
Thr
GCG
Ala
CGG
Arg
310
TCG
Ser
135
GCC
Ala
GAC
Asp
ACG
Thr
ACC
Thr
CCC
Pro
215
CAC
His
TCT
Ser
TTC
Phe
TGC
Cys
CAG
Gln
295
CTG
Leu
GCG
Ala
GAG
Glu
GCC
Ala
GCC
Ala
GGG
Gly
200
CCG
Pro
GTA
Val
GAG
Glu
CTG
Leu
ATC
Ile
280
TGC
Cys
TAC
Tyr
PvuII
CAG_CIG
GAG
Glu
GGC
Gly
GGC
Gly
185
CCC
Pro
CGG
Arg
TAC
Tyr
TTT
Phe
CGG
Arg
265
TTC
Phe
AGC
Ser
GAG
Glu
GGT
Gly
CGC
Arg
140
GAC
Asp
GGC
Gly
155
GGC
Gly
GAC
Asp
170
GAG
Glu
GAG
Glu
GCG
Ala
CAG
Gln
GGC
Gly
ACC
Thr
CCC
Pro
GGC
Gly
CGG
Arg
CAC
His
GGC
Gly
220
SmaI
ACC_CCG_GGC
Thr Pro Gly
TTC
Phe
250
GAC
Asp
GAG
Glu
ACG
Thr
GCC
Ala
GGC
Gly
CAC
His
CCC
Pro
GAG
Glu
TTC
Phe
GCG
Ala
TCG
Ser
300
CAG
Gln
TGC
Cys
315
CGC
Arg
AAC GCG ACC
Asn_A1a_Ihr Gly
GTG
Val
ACG
Thr
GCC
Ala
CCG
Pro
205
GCG
Ala
GAT
Asp
GCT
Ala
GAC
Asp
GCA
Ala
285
CCG
Pro
CCG
Pro
TAC
Tyr
CAG
Gln
GCG
Ala
190
CCG
Pro
CGC
Arg
TCC
Ser
CCC
Pro
TGC
Cys
270
CCG
Pro
TAC
Tyr
GAC
Asp
TTC
Phe
TTG
Leu
175
CGG
Arg
CCC
Pro
TTC
Phe
TTT
Phe
TTC
Phe
255
GCG
Ala
GCC
Ala
CGC
Arg
CCT
Pro
CTG
Leu
160
GCG
Ala
GAC
Asp
CAC
His
CGC
Arg
CTG
Leu
240
TCG
Ser
CTC
Leu
TGC
Cys
TCC
Ser
GCC
Ala
320
GGT
Gly
GCG
Ala
GCG
Ala
GGC
Gly
TTG
Leu
385
GAC
Asp
CCC
Pro
CTC
Leu
CGC
Arg
CAC
His
CCG
Pro
CAC
His
370
GTG
Val
GCT
Ala
ACC
Thr
GCG
Ala
TGG
Trp
CTG
Leu
GCT
Ala
355
GTG
Val
CGC
Arg
CCC
Pro
GGG
Gly
GGA
Gly
435
CCG
Pro
CGT
Arg
340
GCG
Ala
HindIII
CAC
His
325
CCC
Pro
GCC
Ala
GAG
Glu
GCC
Ala
TCC
Ser
GAAJKELIGG
Glu Ala Trp
GCG
Ala
GAG
Glu
CCC
Pro
420
CTG
Leu
GTC
Val
CCA
Pro
405
GCG
Ala
ACC
Thr
390
GGC
Gly
CCC
Pro
TGC GAG GGC
Cys Glu Gly
GGG
Gly
Asp
375
GAC
Asp
CCA
Pro
TGG
Trp
CTT
Leu
360
TAC
Tyr
CAC
His
CCG
Pro
CTT
Leu
TAC
Tyr
AGC
Ser
ACG
Thr
CTC
Leu
GTG
Val
425
GTG
Val
GGC
Gly
ATC
Ile
GCA
Ala
440
GCC
Ala
GCC
Ala
330
GTA
Val
GTC
Val
CTA
Leu
CGC
Arg
ACC
Thr
410
GTG
Val
GCG
Ala
GAC
Asp
TTT
Phe
GTC
Val
CCC
Pro
395
AGC
Ser
CTG
Leu
TAC
Tyr
CTG
Leu
GTG
Val
GTT
Val
380
GAG
Glu
GAG
Glu
GTG
Val
GCG
Ala
GCG
Ala
GTC
Val
TTT
Phe
350
CTG
Leu
365
CAG
Gln
ACT
Thr
TCG
Ser
GCC
Ala
GCA
Ala
CCG
Pro
GCG
Ala
GGC
Gly
GCG
Ala
430
TRANSMEMBRANE HELIX
CTC
Leu
GCC
Ala
GTT
Val
CGG GTG
Arg Val
445
CCC
Pro
335
GAC
Asp
TAC
Tyr
GAC
Asp
GCC
Ala
GGC
Gly
415
CTT
Leu
GTT
Val
GAC
Asp
AAC
Asn
CGT
Arg
GCC
Ala
400
GCG
Ala
GGA
Gly
==a==::n==
TGC
Cys
CGC
Arg
CCC
Pro
465
CCA
Pro
CGC
Arg
450
GTA
Val
GTT
Val
GCA
Ala
TAC
Tyr
AGC
Ser
AGC
Ser
ACC
Thr
GAC
Asp
CAG
Gln
AGC
Ser
GAC
Asp
485
AAG
Lys
TTG
Leu
470
GAA
Glu
CGC
Arg
455
CCG
Pro
TTT
Phe
ACC
Thr
ACC
Thr
TCC
Ser
TAC
Tyr
AAC
Asn
CTC
Leu
GAC
Asp
GAG
Glu
GAC
Asp
490
ATC
Ile
CCG
Pro
475
GAA
Glu
CTC
Leu
460
CTC
Leu
GAC
Asp
AAC
Asn
CCC
Pro
GAC
Asp
GTG
Val
TCT
Ser
TTT
Phe
TTC
Phe
GTG
Val
GCG
Ala
495
GCG
Ala
GGG
Gly
GTG
Val
480
GAT
Asp
GAC
Asp
TAC
Tyr
CCC
Pro
AGG
Arg
545
CCA
Pro
GAC
Asp
GAC
Asp
GCC
Ala
GAC
Asp
GAT
Asp
AGC GAC GAT
Ser Asp Asp
500
CTC GCC GGC
Leu Ala Gly
515
AAC GGC ACG
AsnJߴ_In:
CCG CTT GAA
Pro Leu Glu
TAC ACC GTG
Tyr Thr Val
565
GAC
Asp
GCC
Ala
CGC
Arg
GAC
Asp
550
GTA
Val
GGG
Gly
CCA
Pro
TCG
Ser
535
GAT
Asp
GCA
Ala
CCC
Pro
GAG
Glu
520
AGT
Ser
GCC
Ala
GCG
Ala
GCT
Ala
505
CCA
Pro
CGC
Arg
GCG
Ala
CGA
Arg
AGC
Ser
ACT
Thr
TCT
Ser
CCA
Pro
Leu
570
AAC
Asn
AGC
Ser
GGG
Gly
GCG
Ala
555
AAG
Lys
GCGCCCCCCC CCCCCCGCGC GCTGTGCCGT CTGACGGAAA
ATATAAATGG AGCGCTCACA CAAAGCCTCG TGCGGCTGCT
CGCAGCGTCG TC
SEQ ID NO:2
LENGTH: 20 nucleotides
TYPE:
nucleotide
STRANDEDNESS:
single
ACGTGGTGGT GCCAGTTAGC
SEQ ID NO:3
22 nucleotides
LENGTH:
TYPE:
nucleotide
STRANDEDNESS:
single
ACCAAACTTT GAACCCAGAG CG
CCC
Pro
GGG
Gly
TTC
Phe
540
CGG
Arg
TCC
Ser
GCACCCGCGT GTAGGGCTGC
TCGAAGGCAT GGAGAGTCCA
CCT
Pro
TTT
Phe
525
ACC
Thr
GCG
Ala
510
GCG
Ala
GTT
Val
CCG
Pro
GAT
Asp
CGA
Arg
TGG
Trp
GCC
Ala
ECONI
ATC_£I£_CG£_IAG
Ile Leu Arg
GCC
Ala
GCC
Ala
TTT
Phe
GCA
Ala
560
2027
Claims (1)
- CLAIMS: A mutant of bovine herpesvirus type 1 (BHV—1) having a deletion in the glycoprotein gE—gene, gE being a protein which, in a particular BHV—1 strain, has the amino acid sequence as shown in figure 3A, wherein said deletion allows the mutant to be distinguished serologically from wild—type BHV-1. A BHV—1 mutant according to claim 1 wherein said deletion in the gE—gene has been caused by an attenuation procedure. A BHV—1 mutant according to claim 2 which is Difivac—1 (Institut Pasteur, France, deposit No. 1- 1213). A BHV—1 mutant according to claim 1 wherein said deletion in the gE—gene has been constructed by recombinant DNA techniques. A BHV—1 mutant according to claim 1 which, in addition to said deletion in the gE—gene, has a deletion in the thymidine kinase gene. A BHV—1 mutant according to claim 1 which, in addition to said deletion in the gE—gene, has a deletion in the glycoprotein gI—gene. A BHV—1 mutant according to claim 1 which, in addition to said deletion in the gE—gene, has a deletion in the thymidine kinase gene and a deletion in the gI—gene. A BHV—1 mutant according to claim 1, selectable or selected by a process of discriminating between BHV—1 viruses having an intact gE—gene and BHV—1 viruses having a deletion in the gE—gene, said process comprising the step of examining whether nucleic acid of the virus reacts with gE—specific probes or primers derived from the nucleotide sequence coding for gE. A BHV—l mutant according to claim 1, selectable or selected by a process of discriminating between BHV—l viruses expressing gE and BHV—l viruses having a deletion in the gE—gene, said process comprising the step of examining whether the virus reacts with gE—specific antibodies raised against gE or against peptides derived from the amino acid sequence of gE. A BHV—l mutant according to claim 1 which contains a heterologous gene introduced by recombinant DNA techniques. A BHV—l mutant according to claim 10, said heterologous gene being inserted at the location of the gE—gene and being under the control of regulatory sequences, of the gE—gene or a e.g. heterologous gene, and optionally attached to the part of the gE—gene that codes for a signal peptide. A BHV—1 mutant according to claim 10 which, in addition to a deletion in the gE—gene, has a deletion in the thymidine kinase gene, a deletion in the gI—gene, or both, said heterologous gene being inserted at the location of at least one of said deletions. A BHV—l mutant according to claim 10 which, in addition to a deletion in the gE—gene, has a deletion in the thymidine kinase gene, a deletion in the gI—gene, or both, said heterologous gene being inserted at the location of at least one of said deletions, with the proviso that in the case of a mutant having a double deletion comprising a deletion in the gE—gene and a deletion in the thymidine kinase gene, said heterologous gene is not inserted at the location of the thymidine kinase gene. A BHV—l mutant according to claim 10 which, in addition to a deletion in the gE—gene, has a deletion in the thymidine kinase gene, a deletion in the gI—gene, or both, said heterologous gene being inserted at the location of at least one of said deletions, with the proviso that in the case of a mutant having a double deletion comprising a deletion in the gE—gene and a deletion in the thymidine kinase gene, said heterologous gene is not a bovine respiratory syncytial virus F or N gene. A BHV—l mutant according to claim l0 which, in addition to a deletion in the gE—gene, has a deletion in the thymidine kinase gene, a deletion in the gI—gene, or both, said heterologous gene being inserted at the location of at least one of said deletions, with the proviso that in the case of a mutant having a double deletion comprising a deletion in the gE—gene and a deletion in the thymidine kinase gene, at least two heterologous genes are inserted. A BHV—1 mutant according to anyone of claims 10 to 15 wherein said heterologous gene codes for an immunogenic protein or peptide of another pathogen or codes for a cytokine. A composition comprising recombinant nucleic acid which contains the gE~gene of BHV—1, a gE—specific part of this gE—gene or a gE—specific nucleotide sequence derived from this gE—gene, gE being a protein which, in a particular BHV—1 strain, has the amino acid sequence as shown in figure 3A. A composition according to claim 17 comprising a cloning or expression vector having therein an insertion of recombinant nucleic acid containing the gE—gene of BHV—1, a gE—specific part of this gE—gene or a gE—specific nucleotide sequence derived from this gE-gene. A composition comprising gE of BHV—1, a gE—specific part of this gE, a gE—specific peptide derived from this gE, or a complex of the BHV—1 glycoproteins gE and g1, gE being a protein which, in a particular BHV—1 strain, has the amino acid sequence as shown in figure 3A. A composition comprising an antibody which is specific for gE of BHV—1, a gE—specific part of this gE, a gE—specific peptide derived from this gE, or a complex of the BHV—1 glycoproteins gE and g1 gE being a protein which, in a particular BHV—1 strain, has the amino acid sequence as shown in figure 3A. A composition according to claim 20 comprising a monoclonal antibody that is specific for gE of BHV— 1, a gE—specific part of this gE, a gE—specific peptide derived from this gE, or a complex of the BHV—1 glycoproteins gE and g1. A composition according to claim 20 Comprising a polyclonal antibody which is specific for gE of BHV—1, a gE—specific part of this gE, a gE—specific peptide derived from this gE, or a complex of the BHV—1 glycoproteins gE and g1. A vaccine composition for a vaccination of animals, in particular mammals, more in particular bovines, to protect them against BHV—1, wherein the vaccine composition is a live or an inactivated vaccine comprising a BHV-1 mutant according to anyone of claims 1-16, and a suitable carrier or adjuvant. A vaccine composition for a vaccination of animals, in particular mammals, more in particular bovines, to protect them against a pathogen, wherein the vaccine composition is a live or an inactivated vaccine comprising a BHV-1 mutant according to anyone of claims 1-16 which has a deletion in the gE—gene and contains a heterologous gene which has been introduced by recombinant DNA techniques and codes for an immunogenic protein or peptide of the pathogen, and a suitable carrier or adjuvant. A diagnostic kit for detecting nucleic acid of BHV— 1 in a sample, in particular a biological sample blood cells, such as blood or blood serum, milk, bodily fluids such as tears, lung lavage fluid, nasal fluid, sperm, in particular seminal fluid, saliva, sputum or tissue, in particular nervous tissue, coming from an animal, in particular a mammal, more in particular a bovine, comprising a nucleic acid probe or primer with a nucleotide sequence derived from the gE—gene of BHV—l, gE being a protein which, in a particular BHV—l strain, has the amino acid sequence as shown in figure 3A, and a detection means suitable for a nucleic acid detection assay. A diagnostic kit for detecting antibodies which are specific for BHV—l, in a sample, in particular a biological sample such as blood or blood serum, saliva, sputum, bodily fluid such as tears, lung lavage fluid, nasal fluid, milk or tissue, coming from an animal, in particular a mammal, more in particular a bovine, comprising gE of BHV-l, a gE- specific part of this gE, a gE—specific peptide derived from this gE, or a complex of the BHV—l glycoproteins gE and g1, gE being a protein which, in a particular BHV—1 strain, has the amino acid sequence as shown in figure 3A, and a detection means suitable for an antibody detection assay. A diagnostic kit according to claim 26, which further comprises one or more antibodies which are specific for gE of BHV—l, or a complex of the BHV—l glycoproteins gE and g1. A diagnostic kit for detecting antibodies which are specific for BHV—l, in a sample, in particular a biological sample such as blood or blood serum, saliva, sputum, bodily fluid such as tears, lung lavage fluid, nasal fluid, milk or tissue, coming from an animal, in particular a mammal, more in particular a bovine, comprising an antibody which is specific for gE of BHV-l, or a complex of the BHV—l glycoproteins gE and g1, gE being a protein which, in a particular BHV—l strain, has the amino acid sequence as shown in figure 3A, and a detection means suitable for an antibody detection assay. A diagnostic kit for detecting protein of BHV—l in a sample, in particular a biological sample such as blood or blood serum, blood cells, milk, bodily fluids such as tears, fluid, lung lavage fluid, nasal sperm, in particular seminal fluid, saliva, sputum or tissue, in particular nervous tissue, coming from an animal, in particular a mammal, more in particular a bovine, comprising an antibody which is specific for gE of BHV—l, or a complex of the BHV-l glycoproteins gE and gI, gE being a protein which, in a particular BHV—l strain, has the amino acid sequence as shown in figure 3A, and a detection means suitable for a protein detection assay. A method of determining BHV—l infection of an animal, in particular a mammal, more in particular a bovine, comprising examining a sample coming from the animal, in particular a biological sample such as blood or blood serum, blood cells, sperm, in particular seminal fluid, saliva, sputum, bodily fluid such as tears, lung lavage fluid, nasal fluid, milk or tissue, in particular nervous tissue, for the presence of nucleic acid comprising the gE—gene of BHV—l, or the presence of gE of BHV— l or a complex of the BHV—l glycoproteins gE and gl, or the presence of antibodies which are specific for gE of BHV—l or a complex of the BHV—l glycoproteins gE and g1, gE being a protein which, in a particular BHV—l strain, has the amino acid sequence as shown in figure 3A. A method of determining BHV—l infection of an animal, in particular a mammal, more in particular a bovine, comprising examining a sample coming from the animal, in particular a biological sample such as blood or blood serum, blood cells, sperm, in particular seminal fluid, saliva, sputum, bodily fluid such as tears, nasal fluid, milk, lung lavage fluid, or tissue, in particular nervous tissue, for the presence of nucleic acid comprising the gE—gene of BHV—l, or the presence of gE of BHV— l or a complex of the BHV—1 glycoproteins gE and g1, or the presence of antibodies which are specific for gE of BHV—l or a complex of the BHV-1 glycoproteins gE and g1, gE being a protein which, in a particular BHV—l strain, has the amino acid sequence as shown in figure 3A, the sample to be examined coming from an animal which has been vaccinated with a vaccine preparation according to claim 23. F. R. KELLY & CO., AGENTS FOR THE APPLICANTS
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
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IE1992/2829A IE83654B1 (en) | 1992-11-19 | Bovine herpesvirus type I deletion mutants, vaccines based thereon, diagnostic kits for detection of bovine herpesvirus type I |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
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IE1992/2829A IE83654B1 (en) | 1992-11-19 | Bovine herpesvirus type I deletion mutants, vaccines based thereon, diagnostic kits for detection of bovine herpesvirus type I |
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Publication Number | Publication Date |
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IE922829A1 IE922829A1 (en) | 1994-06-01 |
IE83654B1 true IE83654B1 (en) | 2004-11-03 |
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