Note: Descriptions are shown in the official language in which they were submitted.
The present invention relates to vaccines based on type
1 bovine herpesviruses (BHV-1) which contain modifi-
cations in regions of their genome which code for non-
essential parts of essential proteins. It is possible
with the aid of these vaccines to differentiate immunised
from non-immunised cattle. The invention furthermore
relates to processes for the isolation and preparation of
the modified BHV-1 strains, isolation and preparation of
the modified proteins and peptides.
Infection with type 1 bovine herpesvirus (BHV-1) in
cattle becomes manifest mainly in the organs of the-
respiratory tract and of the genital tract, hut also in
the peripheral and central nervous system. The clinical
pictures induced thereby are described as infectious
bovine rhinotracheitis, infectioua bovine vulvovaginitis
and infectious pustulous balanophosthitis. Besides these,
conjunctivitis, orchitis, endometritis, mastitis, abor-
tions or meningoencephalitis are also observed. following
the initial infection there may be latent persistence of
BHV-1 throughout the life of the infected animal. The
cells of the nervous system, but also the epithelial
tissue, the lympihocytic tissue, macrophages and lympho-
cytes are suggested as sites of virus persistence. As a
consequence of reactivations of these latent infections,
phases with clinical manifestation with virus excretion
alternate with phases with inapparent symptoms with or
without excretion of pathogen. The causes of reactivation
Le A 29 649 - 1 -
zo~~a.9~
are strains on the organism with latent infection, such
as stress, immunosuppression or secondary infection.
Because of the serious economic damage (such as, for
example, rearing losses, decrease in milk yield, reduc-
tion in fertilisation rate, sterility and abortions)
caused world-wide by the disorders associated with BHV-1,
prophylactic control measures have great importance.
Besides the damage caused by the disorders, the indirect
consequences of BHV-1 infection have great importance.
Thus, only very restricted trade nationally and inter-
nationally in cattle suspected of BHV-1 is allowed.
Available for the prophylactic control of disorders
associated with BHV-1 are live vaccines based on attenu-
ated BHV-1 strains, inactivated vaccines and so-called
subunit vaccines based on purified virus proteins. In the
control measures currently in progress for the eradi
cation of BHV-1 or for freeing herds infected with BHV-1
of the disease, arid in livestock export, cattle are
identified as possible virus carriers on the basis of
detecting BHV-1-specific antibodies.
Differentiation of BHV-1-immunised cattle from BHV-1-
infected cattle, which could make measures for eradi-
cation and fre:sing of disease considerably more
effective, is not at present passible.
This type of differentiation of immunised from infected
livestock is possible in other areas. Vaccines for pigs
Le A 28 649 _ 2 -
for the prophylaxis of the clinical mazaifestation of
infection with Aujeszky virus (procine herpesvirus 1)
have already been developed and permit differentiation of
immunised and infected livestock. These vaccines are
based on virus strains which lack, entirely or in parts,
proteins which are not necessary for virus replication,
so-called non-essential virus proteins, or they are based
on individual purified virus proteins. Serological
detection methods for detecting specific antibodies
directed against the virus proteins missing from the
vaccine permit identification of Aujeszky virus-infected
pigs and differentiation from livestock immunised against
this pathogen.
A vaccine described in European Hublished Specification
316 658 is said to permit differentiation of immunised
and infected cattle. A HHV-1 mutant prepared by recom
binant technology, which does not, because of a mutation
(deletion or insertion) in the gene for the non-essential
structural glycoprotein gIII, contain this glycoprotein,
is described.
It is possible to assign particular biological functions
to individual, viral proteins. The absence of protein
components in viruses, such as, far example, of glyco-
proteins, necessarily determines the absence of biologi-
cal functions of these viruses. Thus, the non-essential
structural protein gIII of Aujeszky virus is signifi-
cantly involved in the immune response to Aujeszky virus.
Specifically cellular defence mechanisms, which have a
Le A 28 649 - 3 -
CA 02085191 2002-07-22
23189-7445
predominant role in the immunological r_iefence against
herpesviruses, are directed against the protein gIII of
Aujeszky virus (Zuckermann et al., 199C), J. Virol. 64, 802-
812 ) .
Applied to the BHV-1 system, this means that the
absence of the protein gIII, comparable in function, in
BHV-1 should bring about a reduction. in th.e immunogenicity.
In order to prepare BHV-1 vaccines with high immunogenicity,
the BHV-1 mutants which are employed a~> immunising viruses
must therefore have deletions or other- alterations in
proteins or protein sections which are not the aim of
relevant defence mechani.sm:~ .
According to one aspect of the present invention,
there is provided a use of a strain of BHV-1 for the
preparation of a vaccine against one or more BHV-1
infections which permit dz.f=ferentiation of vaccinated
livestock from field-infected livestock, said strain of BHV-
1 containing one or more modified DNA sequences) encoding
one or more non-essential regions of glycoprotein gIV of BHV
strain Reg. No. I 1204, said one or more non-essential
regions relating to epitopes which correspond to amino acid
positions 310 to 338 in said glycoprote:in gIV of said BHV
strain Reg. No. I 1204.
According to another aspect c~f the present
invention, there is provided the use as described above,
wherein strain Reg. No. I 1204 is emplc.~yed as the BHV-1
strain.
According to still another aspect of the present
invention, there is provided a use of one or more peptides
which are homologous to one or more amino acid sequences
_ 4 _
CA 02085191 2002-07-22
23189-7445
of one or more non-essential regions of= glycoprotein BHV-1
field viruses which are het:erologous too the modified amino
acid sequence positions 310 to 338 in :e=_rological methods
for differentiating cattle which are infected with BHV-1
field viruses from cattle which have been immunized with
vaccines based upon BHV--1 strain Reg. I: 1204.
The present invention now relates to:
1. Use of strains of BHV-1 which, compared with
field viruses, contain, because of a DT~dA sequence which is
modified at one or more points, one or more modified amino-
acid sequences in non-essential regions of essential
proteins for the preparation of vaccines against BHV-1
infections.
2. Use of BHV-1 strains acc°ording to (1), in
which the modifications affect the essential glycoprotein
gIV.
3. Use of BHV-1 strains according to (1), in
which the modifications affect the region of glycoprotein
gIV which projects directly out of the merr~bran.e coat of the
virus.
4a -
~0~~.~~1
4. Use according to (1)-(3), wherein the subtype 3
(BI3V-1.3) is employed as H1HV-1 strain.
5. Use according to (1)-(4), wherein the strain N 569
is employed as HHV-1 strain.
6. Use according to (5), wherein the amino-acid
sequence of the glycoprotein gIV in the strain N 569
has been modified compared with a field virus in the
region of positions 310 to 338.
7. Use of BHV-1 strains according to (1), in which the
amino-acid sequences in the glycoprotein gIV, which--
correspond in the region to the positions 310 to 338
of the glycoprotein gIV of the strain N 569, are
modified.
8. Process for the preparation of BBV-1 strains for use
according to (1)-(7) characterised in that
a-1) the DNA sequence of viral genes for essential
proteins is determined for a BHV-1 strain which
has been isolated from an infected natural host
(for example cattle, pigs, goats) or passaged
in cells, and the amino-acid sequence of the
encoded protein is derived therefrom or
a-2) the amino-acid sequence of viral essential
proteins is determined directly and
Le A 28 649 - 5 -
z~~~.~9~.
b) the amino-acid sequence determined as in a-1)
or a-2) is compared with the amino-acid
sequence of the corresponding essential pro-
teins of BHV-1 field viruses, or
c) immunological methods using BHV-1 specific
antibodies against essential proteins of field
viruses are used to search for altered amino
acid sequences, and the BHZi-1, strains which
are not recognised by one or more antibodies
are selected, and
d) the strains which have amino-acid sequences-
altered by comparison with the field virus and
which axe obtained according to a), b) or c),
and the field virus are tested for antigenicity
in a manner known per se. In this connection,
the amino-acid sequence of the field virus
which corresponds to the altered amino-acid
sequences of the strains according to a), b) or
c) must be involved in the formation of one or
more epitopes which do not occur in the strains
obtained according to a), b) or c). Cattle not
infected with BHV-1 must not contain any
antibodies against amino-acid sequences of
field viruses, which are altered in the strains
obtained according to a), b) or c).
a ) The strains among those identified according to
Le A 28 649 -
d) which are immunogenic in cattle are
selected.
f) As an alternative to a) to c), it is possible
in a manner known per se to alter nucleotide
sequences on the genome of BHV'-1 strains in
such a way that the amino-acid sequences in
non-essential parts of essential proteins are
altered in such a way that one or more epitopes
for cattle are modified or absent by comparison
with the field virus.
9. Use of DNA sequences of BFiV-1 strains which code for
essential proteins with non-essential regions forty
the identification of BHV-1 strains which can be
used according to (1), and for the targeted alte-
ration of DNA sequences, which encode non-essential
regions of essential proteins, of B8V-1 strains for
use according to (1).
10. DNA sequences which code for essential proteins of
BHV-1 strains and are altered in their nucleotide
sequence in such a way that they code for proteins
whose amino-acid sequence is modified in non-
essential regions by comparison with field viruses.
11. DNA sequences which code for the protein gIV of
BHV-1 strains and are altered in their nucleotide
sequence in such a way that the amino-acid sequence
of glycoprotein gIV is modified in non-essential
Ze A 28 6~9 - °7 -
~~8~.~~~
regions by comparison with field viruses.
12. DNA sequence which codes for the essential glyco-
protein gIV of the BAV-1 strain N 569 and is charac-
terised in that it is modified in its nucleotide
sequence in such a way that the amino-acid sequence
of the protein gIV is modified in the region of
positions 310-338 by comparison with field viruses.
13. Process for the preparation of the DNA sequences
according to 11-12, characterised in that the DNA is
isolated from the BHV-1 strains prepared according
to ( 8 ) in a manner known per se, or the genome of
BHV-1 field viruses undergoes molecular cloning in-
vectors in a manner known per se, and the cloned
BHV-1 DNA fragments are altered in their nucleotide
sequence in such a way that, after incorporation
thereof into the genome of BHV-1 strains, they
result in BHV-1 strains which can be used according
to (1), and this modified BHV-1 DNA is isolated from
the vectors in a manner known per se.
14. Use of essential proteins of BHV-1 strains with
modifications in non-essential regions for the
identification of BHV-1 strains which can be used
according to (1), and for the preparation of
vaccines, and for the preparation of BHV-1 strains
with modified amino-acid sequences.
15. Essential proteins of BHV-1 strains Which are
Le A 28 649 - g -
modified in their amino-acid sequence in non-
essential regions by comparison with field viruses.
16. Protein gIV of B~iV-1 strains, whose amino-acid
sequence is modified in non-essential regions by
comparison with field viruses.
17. Protein gIV of the BHV-1 strain N 569, whose amino-
acid sequence is modified in the region of positions
310-338 by comparison with field viruses.
18. Process for the preparation of proteins according to
15-17, characterised in that the protein is isolated
from the BHV-1 strains prepared according to (8) in
a manner known per se, or by cloning and expressing
the genes which code for these proteins in pro' or
eukaryotic expression systems in a manner known per
se.
19. Use of antibodies against non-essential regions of
essential proteins of BBV-1 field viruses for the
identification of BFIV-1 strains which can be used
according to (1).
20. Use according to (19) of antibodies against non-
essential r~sgions of the protein gIV of BFiV-1 field
viruses.
21. Antibodies against non-essential regions, which are
modifiod by comparison with BBV-1 field viruses, of
Le A 28 649 - 9 -
essential proteins of BF3V-1 strains which can be
used according to (1).
22. Process for the preparation of the antibodies
according to ( 21 ) characterised in that animals ( for
example rabbits, mice, goats, sheep) are immunised
with BHV-1 strains which have been prepared accord-
ing to (8), or with essential proteins from BHV-1
strains which are modified in non-essential regions,
or with peptides whose amino-acid sequences cor-
respond to the modified regions non-essential parts
of essential proteins, and the antibodies which are
formed are isolated in a manner known per se.
23. Use of antibodies according to (21) for the identi
fication of BIiV-1 strains which can be used
according to (1).
24. Use of peptides which are homologous with amino-acid
sequences of non-essential regions in essential
proteins o:E BHV-1 field viruses and which are
heterologous with the modified amino-acid sequences
of the non-essential regions in essential proteins
of the BHV-1 strains used according to (1), in
serological methods for differentiating cattle which
are infected with BHV-1 field viruses from cattle
which have keen immunised with vaccines according to
(1).
25. Use of peptides which are homologous with amino-acid
he A 2B 649 - 10
~~5~.9.~
sequences of non-essential regions in the glyco-
protein gIV in BFiV-1 field viruses and which are
heterologous with the modified amino-acid sequences
of the non-essential regions in essential proteins
of the BHV-1 strains used according to (1), in
serological methods for differentiating cattle which
are infected with BHV-1 field viruses from cattle
which have been immunised with vaccines according to
~l).
26. Use of peptides which are homologous with amino-acid
sequences of non-essential regions in the glyco-
protein gIV in BHV-1 field viruses and which are
heterologous with the modified amino-acid sequence-
position 310-338 in the glycoprotein gIV of the BF3V-
1 strain N 569, in serological methods for differen-
tiating cattle which are infected with BHV-I field
viruses from cattle which have beer. immunised with
vaccines based on the BHV-I strain N 569.
The terms mentioned above have the following meanings
BHV means bovine herpesvirus
BHV-1
means bovine herpesvirus of serotype 1 according to
the terminology in "Virus Diseases in Laboratory and
Captive Animals°' F.J. Conraths, H. Ludwig, G. Darai
1988 published by Martinus Nijhoff, Boston.
Le A 28 649 - 11 -
~~Pa~.9~.
BHV-1.3
means bovine herpesvirus of subtype 3 of serotype 1.
Representatives of this subtype were isolated for
the first time in Australia in 1962 as strain N569
(French, 1962, Australian Veterinary journal 38,
216ff) and as strain A663 in Argentina (Carrillo et
al. 1983, Zbl.Vet.Med.B 30, 327ff) where there were
outbreaks of virus-related encephalitis in cattle.
Both Metzler and coworkers (1986, Archives of
Virology 87, 205ff) and Engels and coworkers
(1986187, Virus Research 6, 57ff) described these
isolates, whose DNA restriction enzyme fragment
pattern was uniform and distinguishable by com-
parison with other representatives of BHV type 1, as~
subtype 3 of serotype 1 of BHV. The strain Na67
which was isolated by Bartha and coworkers in
Hungary from a bovine suffering from encephalitis
(Bartha et al., 1969, Acta Veterinariae Academiae
Scientarium Hungaricae ~9, 145ff) showed, according
to investigations by Magyar and coworkers (1989,
Acts Veterinaria Hungaricae 37, 3ff), the typical
BHV-1.3 DNA pattern. Studdert designates this virus
group as "Bovine Encephalitis Herpesvirus (BEHV)"
(Studdert, 1989, Vet.Rec. -, 584).
The repreae~ntatives of subtype 3 play a completely
minor quantitative role compared with the occurrence
of the two other subtypes 1 and 2 of BHV-1 (BHV-1.1
and BHV-1.2)., Subtypes 1.1 and 1.2 differ by a DNA
pattern, which is characteristic of each of them,
Le A 28 649 - 12 -
after treatment of their genomic DNA with certain
restriction enzymes (Engels et al, (1981), Arch.
Virol. 67, 169 ff). Typical representatives which
may be mentioned are the cooper strain for BHV-1.1
and the SchBnboken strain for BHV-1.2.
Field viruses
are type 1 bovine herpesviruses which occur under
natural conditions. Their DNA sequences code for at
least one epitope in non-essential parts of essen-
tial proteins, which is identical in the overwhelm-
ing majority of all variants which occur under
natural conditions and is thus to be designated as
characteristic of BFiV-1 field viruses. The formation
of antibodies in cattle against these (this)
epitope(s) therefore suggests infection with B~iV-1
field viruses.
Essential protein
means one or more proteins which are necessary for
virus replication of BIiV-1 in cell culture. These
proteins arse encoded by the genome of BIiV-1 and may
be a component of the mature virus particle or may
contribute to its replication without becoming a
component of the mature virus particle. Examples of
essential proteins of BHV-1 which may be mentioned
25. are gB and gD (named in accordance with the termi-
nology for ~iSV) (Wyler et al., Infectious Bovine
Rhinotracheitis/Vulvovaginitis, in "Developments in
Veterinary Virology", Vol. °'Herpesvirus Diseases of
Le A 28 6~9 - 13 - .
battle, Horses, and Pigs'°, ed. by Wittmann, ICluwer
Academic Publishers, 1969).
Non-essential regions of essential proteins
designate parts of essential proteins. Alterations
of the amino-acid sequence in these parts are
possible without blocking the function of the
overall protein for virus replication. In connection
with the present invention, it is important that
these non-essential regions are able, on the basis
~ of their amino-acid sequence, to form epitapes which
lead in BHV-1-infected cattle to the formation of
specific antibodies.
Modified amino-acid sequence
means that the amino-acid seguence of an essential
protein of a BHV-1 strain differs in at Least one
non-essential region by comparison with the field
virus in such a way that the epitope present in the
corresponding protein of the field virus in this
(these) regions) is no longer present in the
protein of the virus with the altered amino-acid
sequence. Cattle which become infected with the
virus which contains the modified amino-acid
sequence or receive administration of this virus in
inactivatedl form or parts thereof thus lack specific
antibodies against at least one epitope in the field
virus, which are formed by them after infection with
a field virus.
Lc A 28 649 - 14 -
20~~.~91
The modified amino-acid sequences may appear in
naturally occurring BHV-1 strains, such as, for
example, positions 310 to 338 in the essential
structural protein gIV (gD) of the BHV-1 strain
N569; they may be produced during cell passages of
field viruses or be prepared after identification of
non-essential regions of essential proteins by
altering the DNA sequence in the genome of BHV-1 by
genetic engineering methods:
The modification in the amino-acid sequence of a
non-essential region in an essential protein may be
based on a replacement of one or more aminoacids,
absence of one or more aminoacids, the insertion of--
one or more amino acids or of any combination of the
said possibilities by reason of a DNA sequence of
the virus genome which is modified by comparison
with the field virus. It is crucial that the alte-
ration does not block the function of the essential
protein in virus replication and results in the loss
of at least one epitope, it being perfectly possible
for one or more new epitopes to be produced therein.
Modified DNA sequence
means that the Virus genome of a HBV-1 strain has a
different nucleotide sequence, by comparison with
field viruses, as a consequence of one or more
nucleotide replacements, of a deletion of one or
more nucleotides, of an insertion of one or more
nucleotides or of any combination of these
Le A 28 649 - 15 -
possibilities. The modification results in each case
in the loss of at least one epitope in at least one
non-essential region of an essential virus protein,
it being perfectly possible for one or more new
epitopes to be produced therein although this must
not block the function of the affected virus protein
for virus replication.
Glycoprotein gIV
designates the glycosyl.itic structural protein of
BHV-1, which is designated as homologous gD of HSV
(Wyler et al., Developments in Veterinary Virology;
Vol. Herpesvirus Diseases of Cattle, Horses, and
Pigs; ed. Wittmann; F'luwer Academic Publishers,~-
1989). The nucleotide sequence of the gene for gIV
and its location on the genome are described for the
Sch~nb8ken and Australia BHV-1 strains (Beninga,
diploma thesis in the faculty of biology of the
University of Tiibingen, 1989). The mature glyco-
protein gIV which is incorporated into the extra-
cellular virus particle has, in the typical repre-
sentatives of BHV types 1.1 and 1.2, a relative
molecular weight, determined by SDS polyacrylamide
gel electrophoresis, of about 7lRda (Wyler et al . ,
InfectiouslBovine Rhinotracheitis/Vulvovaginitis, in
"Developments in Veterinary Virology", Vol. "Herpes-
virus Diseases of Cattle, Horses, and Pigs", ed. by
Wittmann, ~Kluwer Academic Publishers, 1989). The
glycoprotein gIV of the BHV-1.3 strains has a
relative molecular weight of about 68 KDa.
Le A 28 649 - 16 -
~~~~1~1
Region which projects directly out of the virus coat of
the virus
means the region in the glycoprotein gIV which
- in
the orientation of the protein from the amino
terminus in the direction of the carboxy terminus
is located shortly before the so-called membrane
domain. Beninga (diploma thesis in the faculty
of
biology of the University of Tiibingen, 1989)
was
able to determine, by sequencing the gene for
gIV of
the BHV-1 Schonboken and Australia strains, the
amino-acid sequence and, from this, the secondary
structure as well as the hydrophilic and hydrophobic
regions of this protein. According to these investi-
gations, a strongly hydrophobic region is to be
found at the amino terminus and is followed by
a
region of about 300 amino acids which has hydro-
philic and hydrophobic regions and is relatively
extensively folded. A linear region predominantly
hydrophilic in character follows in the direction
of
the carboxy terminus. This region with approximately
50 amino acids in the case of the BHV-1 Sch~nb~ken
strain projects directly out of the membrane coat
of
the virus and carries the possible modifications
in
the amino-acid sequence according to 3 ( above
) . This
is followed directly, in the direction of the
carboxy tea.~minus, by a very highly hydrophobic
region which is designated as membrane domain
and
anchors the glycoprotein in the membrane.
Le A 28 649 - 17 -
2~~5~.~~
Natural host
means any animal which can be infected with BBV-1
and replicates this virus. Natural hosts can be
cattle, pigs and goats (Rolls and Mayr, Mikro-
biologie, Infektions- and Seuchenlehre [Micro-
biology, infection and epidemiology textbook]
F. Enke Verlag Stuttgart).
BHV-1 strain passaged in cells
means that a BF~V-1 strain is replicated in
eukaryotic tissue culture cells. This entails the
harvest from a replication on tissue culture cells
or a BHV-1 isolate from an animal, possibly after
storage, being inoculated onto tissue culture cells--
and harvested again after virus replication. The
number of replications on the tissue culture cells
depends in this connection on the aim of the tissue
culture passages. If the virus is to be replicated
to obtain relatively large amounts for analytical or
preparative approaches, a small number, for example
1 to 10 replication cycles suffices. Tf the virus
is to be modified in its biological or biochemical
properties, for example virulence, amino-acid
sequence and/or DNA sequence, by the tissue culture
passages, a larger number of cell passages is
advisable. Thus, fox example, it was possible to
alter the BEV-1 Schonb8ken strain, which was origi-
nally pathogenic for cattle, to apathogenicity by
about 200 cell passages.
Le A 28 649 - 18 -
20~~19~
The occurrence of alterations in the biological or
biochemical properties can be influenced by the
choice of the cells employed for the cell passages.
Cells which, for example, derive from non-natural
hosts of BHV-1, such as, for example, dog kidney
cells, may offer advantages for the selection of,
for example, apathogenic mutants of B~dV-1, which may
result in attenuation of an originally pathogenic
BHV-I strain.
Determination of the DNA sequence
means that the virus strain to be investigated is
replicated, the virus genome is isolated and at
least parts thereof undergo molecular cloning in-
vectora. The clones with viral genes for essential
I5 proteins can be identified by hybridisation using
DNA probes. Examples of DNA probes which can be
employed are DNA fragments or synthetic oligonucleo-
tides with nucleotide sequences of genes for
essential proteins of BHV-1 or of other herpes-
viruses. Nucleotide sequences of BI~V-1 genes, or DNA
fragments with parts of or with complete BHV-1 genes
for essential proteins of BHV-1, for example gD and
gB, which can be employed as DNA probes, are known
from Benin~ga (diploma thesis in the faculty of
biology of the University of Tiibingen, 1989) and
Chase and coworkers (Journal of Tissue Culture
Methods, ~1, 75ff, I988, and J.Gen.Virol. 70,
1561ff, 1989). The identified DNA clones which carry
DNA sequences of the virus genome for non-essential
Le A 28 649 - I9 -
proteins can be used to determine the nucleotide
sequence of the virus genes by the methods known per
se.
To determine the amino-acid sequence directly
means that the amino-acid sequence of one or more
essential BHV-1 proteins is determined by the
biochemical methods which are known per se. The
essential proteins are previously purified from BHV-
1 infected cells or from virus particles by, for
example, immunoaffinity chromatography using known
monoclonal antibodies against the essential virus
proteins such as, for example, gB and gD (Chase and
coworkers, Journal of Tissue Culture Methods, 11,
75ff, 1988; Van Drunen Littel-van den Hurk and
coworkers, Vaccine 8, 358ff, 1990; Fitzpatrick and
coworkers, Virology 176, 145ff, 1990; Duque and
coworkers, Vaccine 7, 513ff, 1989; Marshall and
coworkers, Virology 165, 338ff, 1988).
Alternatively, the proteins to be sequenced can also
be synthesised in eu- or prokaryotic expression
systems and then purified by immunoaffinity
chromatography.
Comparison of than amino-acid sequence
means that the amino-acid sequence of essential
proteins of the investigated BHV-1 strain is com
pared with the amino-acid sequence of the corres
ponding essential proteins of ane or more BHV-1
field vixuses. To do this, the amino-acid sequences
Le A 28 6~9 - 20 -
~08.~.~~~.
to be compared are laid against one another in the
same orientation from the amino terminus to the
carboxy terminus diagrammatically in such a way that
identical amino acids are present at the maximum
number of positions in the sequences to be compared.
Tn order to achieve this it is also perfectly
possible for the individual amino-acid sequences to
be diagrammatically separated and interrupted.
Epitopes in the essential proteins of BIiV-1 field
viruses which do not occur in the essential proteins
of the investigated BBV-1 strain are sought. Regions
of at least 5 consecutive amino acids in the amino-
acid sequence of the essential proteins of the field
virus (or of the field viruses) which do not occur
in the corresponding proteins of the investigated
BFIV-1 strain indicate, for example, possible modifi-
cations of epitopes in the investigated BBV-1
strain.
Specific antibodies
designates sera or serum fractions from animals with
antibodies against parts of essential proteins of
BHV-1 field viruses, or monoclonal antibodies (in
the form o:E hybridoma culture media or ascites from
mice treated with hybridoma cells) against indivi-
dual epitopes of essential proteins of BHV-1 field
viruses. B1HV-1 strains which have a modified amino-
acid sequence in the corresponding regions of their
essential proteins, which these antibodies recognise
~e A 28 649 - 21 -
~0~19~
in BFIV-I field viruses, are not recognised by these
antibodies.
Sera or serum fractions from cattle are preferred.
To prepare these antibodies, animals, for example
cattle, which have no antibodies against HIiV-1 are
immunised with parts of essential proteins of HHV-1
field viruses, where appropriate with the addition
of adjuvants, by administration one or more times.
The sera of the immunised animals can be employed
unpurified os as serum fractions which contain only
antibodies directed against the antigens employed
for the immunisation. Serum fractions of this type
are obtained, for example, by purification by
affinity chromatography.
The cattle can also be immunised with intact HHV-1
field viruses. It is then necessary in every case to
purify the serum fractions from these animals, which
contain antibodies only against individual parts of
essential proteins of BHV-1 field viruses. This can
be carried out by treating the sera with purified
parts of essential proteins from HFfV-1 field viruses
as antigen to adsorb corresponding antibodies. The
adsorbed antibodies are then eluted and collected
(immune-affinity chromatography).
Monoclonal antibodies from, for example, mice which
have been immunised with intact BI~V-1 field viruses
Le ~1 28 649 _ 22 -
2~~~19~.
or purified essential proteins from BHV-1 field
viruses or with parts of essential proteins of BHV-1
field viruses are very particularly preferred. The
immunisation of the animals, fusion of their spleen
cells with myeloma cells and obtaining of the
antibodies takes place according to the methods
known per se for the preparation of monoclonal
antibodies ("Antibodies, a Laboratory Manual";
E. Harlow and D.Lane; Cold Spring Harbor Laboratory;
ZO 1988). If the mice are immunised with intact BHV-1
field viruses it is necessary to select the
hybridoma cultures which form antibodies against
essential proteins of BHV-l field viruses. These
hybridoma cultures can be selected, for example, by-
testing their cell culture supernatants in an ELISA
with essential proteins from BHV-1 field viruses or
parts of these proteins as antigen.
Immunological methods
mean techniques with whose aid parts of essential
proteins of BHV-1 can be investigated using sera,
serum fractions or monoclonal antibodies. (For
review: "Antibodies, a Laboratory Manual"; E.Harlow
and D. Lane; Cold Spring Harbor Laboratory; 1988).
The sera, eaerum fractions or monoclonal antibodies
used in this case recognise parts of essential
proteins from BHV-1 field viruses. BHV-1 strains
whose essential proteins are not recognised by these
sera, serum fractions or monoclonal antibodies,
which thus have different epitopes in essential
Le A 28 649 - 23 -
~~~~1.91
proteins by comparison with field viruses, are
sought. The choice of the suitable techra.ique can be
based on the serum, serum fraction or monoclonal
antibody material available.
An example of a technique which may be mentioned is
immunoblotting (Western blot). This entails the
proteins from BHV-1 being fractionated by the SDS
polyacrylamide gel electrophoresis method known per
se, and the proteins subsequently being transferred
in an electrical field to filter paper, for example
nitrocellulose filter. This filter is then incubated
with sera or serum fractions with antibodies against
parts of essential proteins of BHV-1 field viruses
or with monoclonal antibodies against essential
proteins of BHV-1 field viruses. The binding of
these antibodies to antigens on the filter is
visualised by enzyme-mediated colour reactions known
per se ("Antibodies, a Laboratory Manual'°; E.Harlow
and D.Lane; Cold Spring Harbor Laboratory; 1988).
2o Another example is the enzyme-linked immunosorbent
assay which is known per se and which likewise
visualises i:he binding of antibodies to antigens via
an enzyme-coupled colour reaction, and in which
BHV-1 is employed as antigen (..Antibodies, a
Laboratory r4anual"; E.Harlow and D.Lane; Cold Spring
Harbor Laboratory; 1988).
Another example is the immunofiuorescence test known
Le A 28 649 - 24 -
208~1~~.
per se, which detects antibody binding to an antigen
by fluorescence.
Testing for antigenicity
means that the corresponding amino-acid sequence in
BHV-1 field viruses which was unidentifiable in the
investigated BHV-1 strain is investigated for its
ability to act as antigen, that is to say to induce
the formation of antibodies, in the animal. This can
take place by, for example,
- investigating this amino-acid sequence by assessing
its solubility in water and the prediction of
possible secondary structures for the probability of~
surface exposure of this sequence in the essential
protein of BEiV-1 field viruses.
- administering to animals, preferably to cattle, a
synthetically prepared peptide with the amino-acid
sequence of the BHV-1 field viruses which was
unidentifiable in the investigated BHV-1 strain.
After this immunisation, blood samples are taken
from the iymnunised animals, far example as weekly
intervals, and the sera of these animals are inves-
tigated fox' the content of antibodies against this
peptide.
investigating the sera of animals, preferably of
cattle, which have been infected with BIit1-1 field
viruses for the content of ant~.bodies against the
Le A 28 649 - 25 -
amino-acid sequence in essential proteins of BHV-1
field viruses which was unidentifiable in the
investigated BHIV-1 strain. To do this, for example,
the sera are investigated for their ability to bind
a synthetically prepared peptide with the amino-acid
sequence from BI3V-1 field viruses which was uniden-
tifiable in the investigated BHV-1 strain. A tech-
nique which may be mentioned are the enzyme-linked
immunoadsorbent assay (ELISA) known per se or the
1~ immuno-dot-blot, known per se, with synthetic
peptides as antigen.
Epitope
is a specific binding site for antibodies based on'
an amino-acid sequence or, where appropriate, on a
glycosylated amino-acid sequence.
Nucleotide sequences
are parts of the genomic DNA of BHV-1.
Alterations of nucleotide sequences
comprise alJl methods which are suitable for modify
2~ ing the nucleotide sequences which code for essen
tial proteins from BHV-1. The modification may be
based on
- deletion of one or more nucleotides and/or
- insertion of one or more nueleotides and/or
- replacement of one or more nucleotides.
Le A 28 6~g - 26 -
~0~~~01
A selection of methods for modifying nucleotide
sequences is given in "Molecular Cloning" 2nd
edition, 1989, ed. ,~.Sambrook, E.F.Fritsch,
T.Maniatis, Cold Spring Barbor Laboratory Press. The
modification of the nucleotide sequence is intended
to eliminate epitopes in essential proteins of BHV-1
without in this way blocking the biological function
of this protein for virus replication.
Deletion
means removal of one or more building blocks from a
sequence without replacement and without blocking
the function of the protein.
Substitution
means that a building block is deleted from a
l~ sequence and is replaced by another building block
at the same site without blacking the function of
the protein.
Isolation of DNA
means that 'the DNA is extracted by molecular genetic
methods known per se from a DNA-containing mixture
(for example from purified virus particles of BHV
1) (Virologische Arbeitsmethoden [Methods in
virology research] Vol. III, ed. A.Mayr;
P.A.Bachmann; B.Ma~yr-Bibrack; G.Wittmann; Gustav
Fischer Verlag).
Le A 28 649 - 27 -
2485191
Molecular cloning of genome
(compare point 14 above) means that the genomic DNA
of BgV-1 is isolated and the DNA fragments which
contain genes or DNA sequences of genes for essen-
tial proteins of HHV-1 are inserted into conven
tional DNA vectors (for example bacterial plasmids
such as pHR322, pUClB/19 inter alia) . The identi
fication of DNA fragments from the genome of BHV-1
which contain genes or parts'of genes for essential
proteins can take place
- by the DNA sequences for genes of essential
proteins from HHV-1 being known (for example gI
described in Chase et al., 1989, J.gen.Virol. 70,-
1561-1569) or
- via homologies of genes for essential proteins
from other herpesviruses such as, for example
herpes simplex virus, Aujeszky virus, equine
herpesvirue l and 4, for which the DNA sequences
of their genes for essential proteins are known:
It is possible via DNA/DNA hybridisations of
these DNA sequences with genomia DNA fragments of
H8V-1 to find DNA fragments of the latter with
genes for essential proteins.
A selection for methods for the preparation and
cloning of DNA fragments is given by "Molecular
Cloning" 2nd edition, 1989, ed. J.Sambrook,
E.F.Fritsch, T.Maniatis, Cold Spring Harbor
Ire A 28 649 - 28 -
~o~~~~~
Laboratory Press. These vectors with the HHV-1 DNA
fragments as inserts are used, for example, for
preparing identical copies of the originally iso
lated DNA fragments with genes or DNA sequences of
genes for essential proteins of BBV-1.
Vectors
which can be employed fox the cloning of DNA frag-
ments of the gename of BHV-1 and/or, where appropri-
ate, for the specific alteration of DNA sequences in
genes for essential proteins of BgV-1 and which may
be mentioned are all conventional bacterial or
eukaryotic plasmid vectors (such as, for example,
pBR322, puClB/19 inter alia), bacterial phages (such
as, fox example, lambda, M13 inter alia). possibili-
ties for the insertion of the BHV-1 DNA fragments
into vectors, the replication thereof in host cells
and the reisolation of the DNA inserts are described
in detail in "Molecular Cloning" 2nd edition, 1989,
ed. J.Sambraok, L.F.Fritsch, T.Maniatis, Cold Spring
Harbor Laboratory Press.
Use of essential proteins of BBV-1 strains with
modifications in non-essential regions fox the
identification of HFiV-1 strains
means that a BHV-1 strain which has modified amino
acid sequences in essential proteins and whose
ability far use according to (1) has been
demonstrated is used to identify other BF~V-1 strains
utilisable according to (1).
Le A 28 649 - 2g -
a. The identification of modified amino-acid sequen-
ces also locates non-essential regions in
essential proteins of BH'7-1 whose amino-acid
sequence can be modified without blocking the
biological function of these proteins for virus
replication. This simplifies the search for other
BHV-1 strains with modified amino-acid sequences
in essential proteins, because narrower delimi
tation of the regions to be investigated in their
essential proteins is possible.
b. Knowledge of non-essential regions in essential
proteins of BHV-1 simplifies the preparation of
BHV-1 strains according to (9f) because this.
means that the region which can be modified, via
modifications of the DNA sequence in the genome
of BHV-1, in essential proteins is known.
c. Antibodies prepared against modified amino-acid
sequences in non-essential parts of essential
proteins of BHV-1 strains can be utilised for the
identification of other BHV-1 strains which have
identical modifications.
d. The essential proteins of BHV-1 which have
modified amino-acid sequences can be employed as
immunogenic components in BHiI-1 vaccines. It is
necessary for these proteins to be prepared in
pure form for this purpose. This can take place,
for example, by known biochemical or
Le A 28 649 - 30
immunological purification methods from virus-
containing material or by expression of genes
which code for these proteins in bacterial or
eukaryotic expression systems. After the immuno-
genicity of these proteins has been verified they
can be employed, where appropriate with the
addition of adjuvants, as vaccines for controll-
ing HHV-1 infections.
Tsolation of the proteins
for preparing proteins according to (16) to (18)
comprises the known methods for the .isolation of
virus proteins from virus-containing material.
Examples which may be mentioned are
a. purification of proteins by antibodies which are
specific for the protein to be purified, in, for
example, immune-affinity chromatography
("Antibodies, a Laboratory Manual"; E.Harlow and
D.Lane; Cold Spring Harbor Laboratory; 1988).
b. purification of virus proteins by chromatographic
methods such as, for example, ion exchange
chromatography based on their specific charge
relationships or molecular sieve chromatographies
based on their molecular weights.
Expression of proteins for preparation according to (16)
to (18) in pro- or eukaryotic expression systems
indicates the known methods of molecular genetics
Le A 28 649 - 31 -
for preparing proteins with the aid of cloned genes.
A review of the conventional expression systems and
instructions for handling them is given in
°'Molecular Cloning°' 2nd edition, 1989, ed.
J.Sambrook, E.F.Fritsch, T.Maniatis, Cold Spring
Harbor Laboratory Press. To do this, the DNA frag-
ments from the genome of BHV-1 which contain genes
for the essential proteins must be isolated. These
DNA fragments are inserted into the conventional
ZO expression vectors in such a way that synthesis of
the encoded proteins is possible. Expression systems
which may be mentioned are
a. prokaryotic systems such as, for example, the
lambda gt-11 system for E.Coli,
b. eukaryotic systems such as, for example, yeast
expression systems, viral vector systems such as,
for example, vaccinia systems or baculo-virus
systems.
Serological methods
for differentiating cattle which are infected with
BHV°1 field viruses from cattle which have been
immunised with vaccines according to (1) and/or have
not been infected with BHV-1 comprise investigations
on sera from cattle for their content of antibodies
against the amino-acid sequences in essential
proteins of BHV-1 field viruses which are modified
in the BHV-1 strains employed for preparing vaccines
Le A 28 649
~o~~~o~
according to (1). The detection of antibodies in
sera against the amino-acid sequence as occurs in
BHV-1 field viruses permits the interpretation that
the animals from which these sera have beers obtained
are or have been infected with a BHV-1 field virus.
Animals whose sera do not recognise this amino-acid
sequence from BHV-1 field viruses have possibly been
immunised with a BHV-1 vaccine according to (1)
based on a BHV-1 strain with a modified amino-acid
sequence and, in any case, are not infected with a
BHV-1 field virus.
Methods which can be mentioned for the determination
of antibodies against amino-acid sequences in BHV-1
field viruses is an ELISA. Employed as antigen in
this ELISA is a short-chain, for example chemically
synthesised, peptide with the amino-said sequence
from BHV-1 field viruses which is modified in the
BHV-1 strains according to (1).
If it is additionally intended to investigate
whether the same animals which have no antibodies
against the amino-acid sequence from BHV-1 field
viruses have been immunised with vaccines according
to ( 1 ) , the sera from these animals are investigated
for further BHV-1 specific antibodies, for example
in an ELTSA with complete BHV-1 particles as anti-
gen. Detection of these antibodies, combined with
the abovmentioned ELISA results, proves that the
animals donating these sera have been immunised with
Le A 28 649 -- 33 -
vaccines according to (1).
A~ Identification of BFiV-1 strains with modified amino-
acid se~xuences according to 9 (above)
Replication of the BHV-1 strain, c~enome purification
and~molecular cloning according to 9a 1
All BHV-1 strains are suitable for replication for
identification. Strains of BHV-1 of the subtype 3
(BBV-1.3) are preferred. The BHV~1.3 strain N569
is particularly preferred.
The strain N569 was deposited on 13th April 1992 in
accordance with the Budapest treaty under Reg.No.I 1204
at the Institut Pasteur CNCM.
The viruses were replicated in a conventional way in
tissue cultures of animal cells as primary cells or
permanent cell lines, for example in bovine cells,
monkey cells, pig cells or dog cells, preferably in
bovine kidney cells such as, for example, the
permanent bovine kidney cell MD BK (ATCC CCL22 or
derivatives thereof) or the primary bovine kidney
cell EBK or monkey kidney cells such as the perms--
nent monkey kidney cell Vero (ATCC CRL1586, CRL1587
or derivatives thereof) or in pig kidney cells such
as the permanent pig kidney cell PK15 (ATCC CCL33 or
derivatives thereof) or in dog kidney cells such as
Le A 28 649 - 34 -
20~~1~.~
the permanent dog kidney cell MDCK (ATCC CCL34 or
derivatives thereof).
Replication takes place in a manner known per se in
stationary, roller or carrier cultures in the form
of closed cell populations or in suspension cul-
tures. The replication media employed for the cells
are all cell culture media known per se, for example
described in the product catalogue of Flow Labora-
tories GmbH, Fost 1243, 5309 Meckenheim, such as, in
particular, the minimal essential medium (MEM) which
contains as essential components amino acids,
vitamins, salts and carbohydrates, completed with
buffer substances such as, for example, sodium
bicarbonate or (hydroxyethylpiperazine-N-2-ethane-
sulphonic acid (Hepes) and, where appropriate,
animal sera such as, for example, sera from cattle,
horses or their fetuses. The use of fetal calf serum
in a concentration of 1-30% by volume, preferably 2-
10% by volume, is particularly preferred.
The cells and cell lawns used for replication of
these viruses are grown in a conventional manner
virtually to confluence or to optimal cell density.
Before infection thereof with viruses, the cell
growth medium is preferably removed and the cells
are preferably washed with virus replication medium.
Employed as virus replication media are, all cell
culture media known per se, such as, in particular,
the abovmentioned MEM. This is followed by infection
Le ~ 28 649 - 35 -
with a virus suspension. In the virus suspension,
the virus i~ diluted in the virus replication medium
in such a way that infection takes place with a MCI
(~ multiplicity of infection corresponds to
infectious virus particles on existing cells) of
0.01-50, preferably 0.10-10.
The viruses are replicated with or without the
addition of animal sera. In the case where serum is
employed, the latter is added to the replication
medium in a concentration of 1-30$ by volume,
preferably 2-10~ by volume.
Infection and virus replication are carried out at-
temperatures between room temperature and 40°C,
preferably between 32 and 39°C, particularly prefer-
ably at 37°C far several days, preferably until the
infected cells have been completely destroyed.
The virus-containing medium from the infected cells
is worked up further, for example by removing the
cell detritus by filtration with pore sizes of, for
example, 0.1-0.45 ~m and/or centrifugation at up to
10,000 g.
Filtrate or centrifugation supernatant are used for
virus concentration and purification. For this,
filtrate or supernatant are subjected to high-speed
centrifugation until the virus particles have
sedimented. It is possible, whets appropriate, to
Le A 28 649 - 36 -
follow with other purification steps by, for
example, centrifugation in a density gradient.
The genome of the viruses which have been replicated
and purified as described above is isolated and
purified.
The extraction of native viral DNA is preferably
carried out by treating the purified virions with
aqueous solutions of detergents and proteases.
Detergents which may be mentioned are anionic,
cationic, amphoteric, non-ionic detergents. Ionic
detergents axe preferably employed. Sodium dodecyl
sulphate, sodium lauryl sulphate are particularly
preferred.
Prateases which may be mentioned are,'all proteases
which operate in the presence of detergent, such as,
for example, proteinase K and Pronase. Proteinase K
may be mentioned as preferred.
Detergents are employed in concentrations of 0.1-10~
by volume, preferably 0.5-3$ by volume.
Proteases are employed in concentrations of
0.01-10 mg per ml of virus lysate, preferably
0.05-0.5 mg per m1 of virus lysate.
Operations are preferably carried out in aqueous
Le A 28 649 -
205191
buffer solution in the presence of DNase inhibitors.
Buffer substances which may be mentioned are: salts
of weak acids with strong bases such as, for
example, tris(hydroxymethylaminomethane), salts of
strong acids with weak bases such as, for example,
primary phosphates or mixtures thereof.
The following buffer system may be mentioned as
preferred: tris(hydroxymethylaminomethane).
The buffer substances or buffer systems are employed
in concentrations which ensure pS values at which
the DNA is not denatured. pH values of 5-9, particu-
larly preferably 6-8.5, very particularly preferably
7-8, are preferred, and particular mention may be
made of operation in the neutral range.
Examples of DNase inhibitors are ethylenediamino-
tetraacetic acid in concentrations of 0.1-10 mMol,
preferably about 1 mM.
The lipophilic constituents of the virus lysate are
subsequently extracted. Used as extractants are
solvents such as phenol, chlorofos~a, isoamyl alcohol
,.....w
... or mixtures thereof. A mixture of phenol and
chloroform/isoamyl alcohol is preferably employed
initially, with the extraction taking place in one
or more stages:
Preferably employed in the last stage of the
~g A 28 649 - 38 -
zo~~~o~
extraction is chloroform/isoamyl alcohol.
Alternatively, it is possible to employ first phenol
and subsequently chloroformlisoamyl alcohol.
Other methods for the isolation of the virus DNA
are, for exaanple, centrifugation of a virus lysate
in a CsCl. density gradient or in gel electrophoresis
(Sharp et al. Biochem. 1973 (I2) pages 3055-3063).
The extraction of nucleic acids is described in
'°Molecular Cloning'°, A Laboratory Manual,
2nd Edition 1989, ed. ~'. Sambrook, E.F. Fritsch and
T. Maniatis, Cold Spring Harbor Laboratory Press.
The DNA extracted in this way is preferably precipi-
tated from the aqueous solution with, for example,
alcohol, preferably with ethanol or isopropanol and
with the addition of monovalent salts such as, for
example, alkali metal chlorides or acetates, prefer-
ably lithium chloride, sodium chloride or sodium
acetate, potassium acetate.
The concentration of alcohol in this case is between
40 and 100$ by volume, preferably between 60 and 80$
by volume, particularly preferably at about 70$ by
volume.
The chlorieie or acetate concentration is between
0.01 or 1 molar, preferably between 0.1 and 0.8
molar, zf LiCl is employed, its concentration is
Le A 28 649 - 3g -
between 0.1 and 1 molar, preferably between 0.4 and
0.8 molar.
Methods for the precipitation of nucleic acids are
described in detailed in °'Molecular Cloning° loc.
cit.. The precipitated DNA is isolated from the
aqueous suspension by, for example, centrifugation,
preferably washed with alcohol, for example 70~ by
volume ethanol, and finally resolubilised in aqueous
buffer solution.
A buffer substance which may be mentianed is tris-
(hydroxymethyl)aminomethane in concentrations of
1-100 mM, preferably 10-SO mM. Preferred pFi values
are 6-8.5, particularly preferably 7-8.
Examples of further additives which may be mentioned
1S are EDTA (ethylenediaminotetraacetic acid) in
concentration of 0.1 to 10 mM, preferably 1 to
10 mM.
Alternatively, the precipitated DNA can also be
resolubilised in 0.1 x SSC buffer (Molecular Clon-
ing, loc. cit.) or in ammonium carbonate buffer.
The viral DNA purified in this way is treated with
restriction enzyme in accordance with the manu
facturer's instruction. Suitable restriction enzymes
are those which recognise at least one cleavage site
specific for them on the virus genome.
Le A 28 649 - 40 -
The genome fragments produced in this way are
isolated, after the electrophoretic fractionation
thereof, fox example, from the separating gel (for
example agarose), and the fragment which contains
the DNA sequence to be investigated is inserted into
a plasmid or a phage vector.
Suitable methods for the fractionation of the DNA
fragments are electrophoretic and chromatographic
methods.
Gel filtration may be mentioned among the chromato-
graphic methods.
Supports which may be mentioned in the electro-
phoretic methods are agarose or polyacrylamide.
Examples of electrophoresis buffers which may be
mentioned are ethylenediaminetetraacetic acid,
phosphate/buffer (EPP) tris(hydroxymethyl)amino-
methane borate-ethylenediaminetetraacetic acid
buffer (TBE) which has the following composition:
tris 10-100 mM, preferably 40-90 ml~, particu-
larly preferably 80-90 mM,
Boric acid 10-100 mM, preferably 40-90 mM, particu
larly preferably 80-90 mM,
EDTA 1-10 mM, preferably 1-2.5 mM
pH 7-9, preferably 8-8.5
Le A 28 649 - 41 -
2085191
or
tris-(hydroxymethyl)aminomethane acetate-ethylenedi-
aminetetraacetic acid buffer (TAE) which has the
following composition:
tris 10-100 mM, preferably 30-90 mM,
particularly preferably 40 mM,
Sodium acetate l-100 mM, preferably 5-50 mM,
EDTA 1-lO mM, preferably 1-2.5
pH 7-9, preferably 7.5-8.5.
A detailed list and description of electrophoresis
buffers is described in
Current Protocols in Molecular Biology 1987-1988,
published by Wiley-Interscience, 1987
- A Practical Guide to Molecular Cloning, B,
15, perbal, 2nd edition published by Wiley-Inter
science, 1988
- Molecular Cloning, loc. cit.
Virologische Arbeitsmethoden (Methods in Virology
Research), Volume III Gustav Fischer Verlag,
1989.
The procedure for the method is described in
"Molecular Cloning" loc. cit., or in Virolog.
Le A 28 649 - 42 -
~~8~~9~.
Arbeitsmethoden Volume loc. cit.,
The DNA fragment to be investigated is isolated from
the support for example by electroelution of the
support region containing the fragment. Altern-
atively by low-melting agarose method (Molecular
cloning loc. cit.) or by adsorption of the DNA
fragment on to glass surfaces (Gene-cleans method).
For the insertion of the DNA fragment, double-
stranded plasmid or phage vector DNA molecules are
treated with restriction enzymes so that ends
suitable far the insertion are produced.
Examples of plasmids used are pAT153, pACYC184,
pUClB/19, pBR322, pSP64/65.
Used as phage vectors are lambda phage variants such
as, fox example, -ZAP, -gtl0/11 or phage M13mp18/19.
The restriction enzymes which can be employed are
known per se, for example from Gene volume 92 (1989)
Elsevier Science Publishers BV Amsterdam.
The plasmidi treated with restriction enzyme, or the
phage vector is mixed with an excess of the DNA
fragment to be inserted, for example approximately
in the ratio 5 to 1, and treated with DNA ligases in
order to bond the DNA fragment end-to-end covalently
into the vector.
Le A 28 649 - 43 -
Ligases are enzymes which are able to loin two DNA
- molecules via 3'-OH-5' radicals.
The ligation mixture is, for replication of the
plasmids or phages, introduced into pro- or
eukaryotic cells, preferably into bacteria.
Cells or bacteria are propagated.
Examples of bacteria used are Escherichia coli
strain K-12 and its derivatives, for example
K 12-600 (Molecular cloning loc. cit.).
The preparation of the ligation mixture and of the
bacterial culture is carried out in a manner known
per se as described in Molecular Cloning loc. cit..
The bacteria which contain plasmids with inserted
foreign DNA, or phages with foreign DNA, are
selected.
Determinations of the DNA seguence of viral genes
az~d deduction of the amino-acid sequences resulting
from the sequence determined of the correspondinct
proteins accordincLto 8 a-1
The nucleotide sequence of the cloned virus DNA
fragments prepared above, or subclones thereof are
determined by the conventional methods as are
described, for example, in Molecular Cloning, loc.
Le A 28 649 -
~~~~19~.
cit.; A Practical Guide to Molecular Cloning, ed. B.
Perbal, A Wiley-Interscience Publication or Viro
lagische Arbeitsmethoden volume III, ed. A. Mayr,
P.A. Bachmann, B. Mayr-Bibrack and G. Wittmann,
Gustav Fischer Verlag.
The amino-acid sequences of the viral proteins are
then determined from the nucleotide sequences via
the genetic code known per se (Biochemie (Bio-
chemistryy, A.L. Lehninger Verlag Chemie 1977).
Direct amino-acid sequencing of viral ,proteins
according to 8 a-2
As an alternative to the determination of the amino-
acid sequence by D~tA sequencing, the sequencing of
the amino-acid building blocks of a protein can also
be carried out directly on the purified protein. A
detailed description of various methods is described
in Virologischen Arbeitsmethoden, volume III, ed.
A. Mayr, P.A. Bachmann, H. Mayr-Bibrack and
G. Wittmann, Gustav Fischer Verlag.
Identification of altered amino-acid- seguences in
viral proteins according to 8b
Altered regions in proteins are identified by
comparing the amino-acid sequence of the inveati-
gated BHV-1 strain with the corresponding amino-acid
sequence of other BHV-1 strains and/or isolates or
Le A 28 649 - 45 -
field viruses. In this connection, an altered region
is regarded as suitable when at least one
epitope is lost by the alteration, for
example when the amino-acid sequence differs in at
least 5 consecutive amino acids from the comparison
strain. It is possible that one or more new epitopes
are produced by th~ alteration.
Immunoloaical method for the identification of BHV 1
strains with altered regions in virus proteins
according to 8 c
As an alternative to DNA sequencing or direct amino-
acid sequencing, it is possible for altered regions
in virus proteins to be identified by poly- or~
monoclonal antibodies which specifically recognise
only parts of a single protein of BHV-1 field
viruses. For this purpose, the ability of these
antibodies to bind to the BHV-1 strain to be invest-
igated is tested. Examples of methods which may be
mentioned for this are:
immunoflorescence test, ELISA, immunoblotting. If,
in contrast to BHV-1 field viruses, the antibody
does not bind to the BHV-1 strain to be investi
gated, then the latter has an altered amino-acid
sequence in the part of the protein for which the
antibody iav specific.
I~e A 2g 649 - 46 -
~~~~1~3.
B. Testina~ for antiuenicity according to 8d
B. ~ 1. Theoretical evaluation of the antictenicitv of an
amino-acid seguence
The physicochemical nature of an amino-acid sequence
(for example the extent of the hydrophilic or hydro
phobic behaviour) provides information on a possible
antigenicity of this amino-acid sequence. It is
possible in this way, using conventional computer
programs, to identify potentially antigenic
sequences.
B.2 Investigations of the antigenicity in vivo
The peptide with the amino-said sequence of BHV-1
field viruses which corresponds to that identified
as altered in the BHV-1 strain investigated is
chemically synthesised or biochemically purified.
This peptide is tested for antigenicity in the
animal, preferably in cattle. If the peptide is in
the form of a hapten, it is advisable to couple this
peptide to a carrier before administration. Carriers
which may be mentioned are bovine serum albumin,
ovalbumin oz: KLH (keyhole limpet haemocyanine). The
method of linking the synthetic peptide to a carrier
is carried out by the methods known per se.
The animals receive single or multiple parenteral or
enteral, preferably parenteral, administration of
Le A 28 649 - 47 -
2085191
the peptide. It is preferable for various dosages of
the peptide to be administered to various groups of
__animals. Dosages which may be mentioned for the
peptide are 0.01 ~g to 100 mg, preferably 0.1 ~g to
1 mg per administration. Serum samples are obtained
from the animals at weekly to monthly intervals and
are investigated for antibodies against the peptide
administered: Methods which may be mentioned for
investigating for antibodies are the conventional
immunological test methods such as, for example,
virus neutralisation tests, enzyme-linked immuno-
assays or immunoblots. Preferably employed are
enzyme-linked imunosorbent assays (ELISA) in which
the peptide administered, the protein from which the-
peptide derives, or intact or defective HHV-1
particles are employed as antigen.
The administered peptide can be -designated as an
antigen when animals measurably form antibodies
after administration of the peptide. This provides
information on the suitability of the investigated
strain as immunising strain according to l (above).
The crucial point is whether the appropriate amino
acid sequence, which has bean identified ae modified
in the investigated HHV-1 strain, in the field virus
is recognised as antigen in cattle.
For this purpose, cattle are infected with a HHV-1
whose amino-acid sequence is not altered and, after
Le A 28 649 - 48 -
2Q~~1~~
the infection, investigated for the formation of
antibodies. Serum samples are taken from the animals
at weekly or monthly intervals and tested iaununo-
logically. If, after the infection, the animals form
antibodies against the amino-acid sequence of the
BHV-1 employed for the infection, corresponding to
that sequence which was identified as modified in
the BHV-1 strain investigated, then the antigenicity
of the unmodified sequence is regarded as proven. An
immunological detection method which may be men
tioned for the deterzninatian of the antibodies
against the unmodified amino-acid sequence is an
ELISA in which a peptide with the complete or with
parts of the unmodified amino-acid sequence is used
as antigen.
Cattle which are infected one or more times with the
investigated BHV-1 strain which contains the modi-
fied amino-acid sequence, and/or receive admini-
stration of the latter as inactivated virus, must
not form any antibodies against the amino-acid
sequence of field viruses which corresponds to that
which has been identified as modified in the B~iV-1
strain inve:atigated.
B.3. Investigation of bovine sera with known BHV-1
antibody status
Bovine sera whose content of BHV-1-specific
antibodies is known are investigated for the content
Le A 28 649 - 4g -
2085191
of antibodies against the complete or parts of the
corresponding amino-acid sequence of BHV-1 field
- viruses, which corresponds to that sequence which
has been identified as altered in the BHV-1 strain
investigated.
An investigated HHV-1 strain with altered amino-acid
sequence can be employed as immunising strain
according to 1) according to the invention when
1) antibodies against the unmodified amino-acid
sequence are also detectable in bovine sera
which contain antibodies against BHV-1, deter-
mined, for example, in the virus neutralisation
test or in the ELISA based on BHV-1 as antigen,
and
2) sera from cattle which contain no detectable
antibodies against BHV-1 also contain no
detectable antibodies against the unmodified
amino-acid sequence.
C. Testina for immunoge c'ryr accordina to 8e
Identification of a BHV-1 strain which is suitable
according to the invention as virus strain of a
BHV-1 marker vaccine includes testing for immuno-
genicity of this strain.
For this purpose, the investigated BHV-1 strain is
Le A 28 649 - 50 -
administered to cattle either as virus capable of
replication (live vaccine) or else as inactivated
virus, in both cases possibly formulated with an
adjuvant. Administration can take place, as
required, intramuscularl
y, subcutaneously, intra-
tracheally, intranasally, intravaginally, intra-
preputially or into the conjunctiva) sac.
The success of immunisation can
- be tested serologically via the induction of
BI3~l-1-specific antibodies after immunisation,
determined via neutralisation test or ELISA, or
-- be determined by an experimental infection test
with virulent BH'V-1. This entails comparison of
the clinical manifestation between immunised
and non-immunised cattle after a BHV-1 infec
tion produced experimentally.
Suitable strains prevent or reduce distinctly the
clinical manifestation of BFiV-1 infection after
immunisation of cattle once or twice.
D. Genetic engineering preparation according to 8f of
BFiV-1 strains for use according to 1
Le A 28 f49 - 51 -
2485191
D.1 identification of non essential regions in essential
proteins of 8HV-1
Identification of regions which are non-essential
fox virus replication in essential proteins can take
place by
a) identification of modified amino-acid sequences in
existent eHV-1 strains. As described above, BHV-1
strains which have modified amino-acid sequences are
identified by DNA or amino-acid analyses. After
identification, the corresponding genome sequences
undergo molecular cloning, if this has not already
taken place.
b) Modification of genome sequences which code for
proteins, for example by replacement of nucleotides,
deletion of nucleotides or insertion of nucleotides,
and subsequent examination of the significance of
the modification for the ability of the modified
virus to replicate, in which case the investigated
genome sequence and the amino-acid sequence
resulting there from are to be regarded as non-
essential for virus replication when an alteration
in the amino-acid sequence does not prevent
replication of the modified virus.
Le A 28 649 - 52 -
208191
D.2 ~tection of the antiaenici~, of the identi :ed
non-essential r~aions for cattle after in1°ection
with BHV-1
a ) Theoretical evaluation of the antiyenicit3r of
an amino-acid seouence
Theoretical evaluation of the amino-acid
sequence identified in D.l takes place as
described above
b) ~vestiaation of the antigenicityWin vivo
After chemical synthesis of the peptide with--
the corresponding amino-acid sequence of 8aV-1
which corresponds to that sequence which has
been identified as non-essential for virus
replication, or after biochemical purification
of this peptide from HHV-1, this peptide is
tested for antigenicity in the animal, prefer-
ably in cattle. The besting in the animal takes
place as described above.
The crucial point is that the amino-acid sequence
which has been identified in BHV-1 as non-essential
is recognised as antigen after an infection with
8HV-1 field virus:
F'or this purpose, cattle are infected with 8HV-1
and, after the infection, investigated for the
Le A 28 ø49 - 53 -
zo~~~.~~
formation of antibodies. For this purpose, serum
samples are obtained from the animals in wee7cly or
monthly intervals and tested immunologically. If,
after the infection, the animals form antibodies
against the amino-acid sequence, identified above,
in BFiV-1 field viruses, then the antigenicity of
this sequence is regarded as proven. An immuno-
logical detection method which may be mentioned for
the determination of antibodies against this amino-
acid sequence is an ELISA which has as antigen this
amino-acid sequence from B~-1 field viruses as
peptide. The pegtide can, for example, be chemically
synthesised or biochemically purified from BFiV-1
field viruses.
Cattle which are infected with BHV-1 or contain the
latter as inactivated antigen do not develop, even
after multiple administration, any antibodies
against the amino-said sequence identified in BHV-1
as non-essential.
D.3 Preparations of a BBV-1 mutant by alteration of the
identified amino-acid seguence by insertion, substitution
or deletion
The identified non-essential region in BHV-1 can be
altered by deletion, insertion or substitution. For
this purpoe;e, the genome fragments which have the
DNA sequence coding for this non-essential amino-
acid sequence undergo molecular cloning.
Le A 28 649 - 54 -
2085.~9.~
Subsequently, the DNA sequences which code for the
non-essential amino-acid sequences are removed,
partly or fox their entire length, from these DNA
sequences, which are preferably cloned in vectors,
by the conventional methods of molecular genetics
and/or, where appropriate, a different nucleotide
sequence is inserted. If only parts of a protein
essential for virus replication are removed, this
deletion must not block the function of the protein
for virus replication. The effect of the alteration
in the amino-acid sequence on virus replication is
checked in tissue culture. BFiV-1 strains whose
amino-acid sequence have been altered in nonessen-
tial parts of essential proteins and which are to be..
employed according to (1) must be able to replicate
in tissue culture.
The inserted nucleotide sequence must likewise not
block the biological function of the corresponding
protein into which it has been inserted, because it
is a protein essential for virus replication, and it
can code for an amino-sequence by means of which
identification of the virus altered in this way is
possible. 3~he DNA sequence altered in this way by
deletion and/or insertion can be recombined, for
example by cotransfection, with the genome of HHV-1
in order t:o obtain recombinant HF3V-1 which has
deletion of the non-essential amino-acid sequence in
whole or in parts or contains in place of the latter
a new, altered amino-acid sequence.
Le A 2g 649 - 55 -
D.4. Establishment of the correlation of the occurrence
- of antibodies against BHV-1 and against the identi
fied amino-acid seauence
a) Investication of bovine sera with known BHV-1
antibody status
Bovine sera whose content of BHV-1-specific
antibodies is known are investigated for the
content of antibodies against the amino-acid
sequence identified above. A method which can
be employed is the abovementioned ELISA based
on a peptide with the amino-acid sequence
identified above as. antigen.
An amino-acid sequence identified as non
essential can be employed as BIiV-1 vaccine
marker according to the invention, when
1) antibodies against the identified amino-
acid sequence can also be detected in
bovine sera which contain antibodies
against BHV-1, determined, for example, in
a neutralisation test or in an ELISA based
on BFiV-1 as antigen, and
2) sera from cattle which contain no detect-
able antibodies against BHV-1 also contain
no detectable antibodies against the
Le A 28 649 - 56 -
identified amino-acid sequence.
- Tt is possible to employ as test once again the
ELTSA described above and based on a peptide
with the identified amino-acid sequence as
antigen.
D.5. Testincr for immunogenicitv
The testing is carried out, as described above.
I. Material and methods
1. Cell and virus material
A perananent bovine kidney cell (MDB~C cell) was used
as cell system.
The following BHV-1 strains were employed in the
investigations:
BHV-1.1 Schleswig-Holstein (SH)
Z5 - BHV-1.2a SchonbSken (SB)
- BHV-1.2b Australia 12 (Ausl2)
- BHA-1.3 N569
SH, SB and Ausl2 strains were used as examples of
BHV-1 field viruses.
Le A 28 649 - 57 -
2085191
2. Cell growth and virus replication
For cell growth, 100 ml of MDBK cells were inocu-
lated with 50x10' cells/ml per Roux dish ( 175 cm2)
and grown in E-MEM + 0.85 g of bicarbonate/litre
(Mayr et al. (1974). Virologische Arbeitsmethoden
volume II. Gustav Fischer Verlag, Stuttgart) + 10%
FCS (foetal calf serum).
For virus replication, 3-5 days after sell inocu-
lation the culture medium was removed from the
confluent cell lawn (about 500x10 cells/Roux dish)
and replaced by E-MEM + 2:0 g of bicarbonate/litre
(Mayr et al. (1974): Virologische Arbeitsmethoden~.
volume II. Gustav Fischer Verlag, Stuttgart). Virus
infection took place with a multiplicity of infec-
tion (MOI) of 0.01 to 0.5: The virus was harvested
3-4 days after virus inoculation, at 100% cpE
(cytopathogenic effect):
3. Determination of the virus titre
96-Well plates (Nunc) with a confluent MDBK cell
lA~ were-used for determination of the titre of
virus suspensions. Serial dilutions in 1og10 steps
10-1 to 10-~ of the virus, prepared in E-MEM + 2.0 g
of bicarbonate, were applied to the cell lawns at
200 ~1/well. 8-Fold determination was carried out
for the titration: The virus titre obtained was
calculated by the method of Spearman and K~Irber
Le A 28 649 - 58 -
(Mayr et al. (1974). Virologische Arbeitsmethoden
volume I. Gustav Fischer Verlag, Stuttgart).
4. Visualisation of the BHV--1 structural proteins
For the visualisation of viral proteins, confluent
MDBK cells were infected and metabolically labelled
with 35S-methionine from 6 to 20 h after infection.
The viral gIV was precipitated from infected cells
with the aid of a monospecific anti._gIy serum,
fractionated in a 10~ SDS polyacrylamide gel via the
method of L~mmli and, after fluorography, visualised
by autoradiography. In this design of experiment,
gIV was already predominantly glycosylated. Only
"mature" forms of gIV were precipitated.
5. Purification of viral DNA
Roux dishes with a confluent MDBK cell lawn were
inoculated with virus and incubated at t37°C for 3-4
days. At 100 cpF, the complete virus suspension was
centrifuged at 5000 xg for 20 minutes, the resulting
pellet was resuspended in PBS (140 mM NaCI; 2.7 mM
KC1; 6.5 mM NaiHP04; 0.7 mM CaCl2; 0.5 mM MgClZi
1.5 mM KI~zPiDw) and again spun down ( 10000 xg;
20 minutes). The supernatants were combined and then
centrifuged at 100000 xg for 1 h and the virus
pellet was resuspended in 100 ~1 overnight. The
virus pellet was then resuspended ad 5 ml of PBS
with 5 mM MgCl2, homogenised, subsequently mixed with
Le A 28 649 - 5~ ..
20~~~.9.~
DNAseI (final concentration 100 ~g/ml) and incubated
at +37°C for 1 h. After the DNAse treatment the
_ va.rus particles were centrifuged through a 15%
strength sucrose cushion at 100000 xg for 1 h.
Subsequently the pellet was resuspended in 1.8 ml of
20 mM tris buffer pH 8.0 and, after addition of
200 ~,1 of 20% strength sarcosyl, incubated at +56°C
for 1 h. The viral DNA was centrifuged in a CsCl
equilibrium gradient (5.7 mM CsCIZp 10 mM tris pH 7;
100 mM EDTA) in a fixed angle rotor at 300000 xg at
20°C for 48 h, the gradient was fractionated and
aliquots were investigated for the DNA content in a
0.6% agarose gel. DNA-containing fractions were
combined, again centrifuged in a CsCI equilibrium
gradient and dialysed against 20 mM tris buffer
pH 8Ø The DNA concentration was determined in a
photometer at 260 nm.
6. Cloning arid sequencing of gIV
6.1 Cloning
The purified viral DNA from the BHV-1 SB strain was
restricted with HindIII (Boehringer-Mannheim, in
accordance with the manufacturer's instructions).
Subsequently the resulting DNA fragments were
separated br~r electrophoresis in a 0.6% agarose gel
in TA buffer (33 mM tris, 66 mM potassium acetate,
10 mM MG acetate, pH 7.9 with glacial acetic acid,
0.1 mg/ml BSA, 0.5 mM DDT), and the HindIII L
Le A 28 649 - 6p -
~~8~~.~~
fragment with 6.6 kbp was electroeluted (Sambrook et
al. (1989). Molecular Cloning. Cold Spring Harbor
Laboratory Press.). Using T4 ligase, the HindIII L
fragment was inserted into the HindIII cleavage site
of the vector pUCl2. For this purpose, the DNA of
the vector was treated with HindIII, recircular-
isation was prevented by eliminating the
5'-phosphate group using alkaline phosphatase, and
the linearised DNA of the vector pUCl2 was incubated
with the isolated HindIII fragment and T4 ligase.
In the case of the Ausl2 strain, the 8.0 kbp HindIII
DNA fragment which cross-hybridises with the HindIII
L fragment of the SB strain was isolated in the-
manner described above and likewise incubated with
the linearised and HindIII-treated DNA of the vector
pUCl2 and the T4 ligase.
The plasmids were subsequently transvected into
B.coli 0600 (Sambrook et al. (1989). Molecular
Cloning. Cold Spring Harbor Laboratory Press.) and
bacterial clones with recombinant plasmids were
selected on the basis of their ampicillin resistance
and lack of lacZ activity (Sambrook et al. (1989).
Molecular Cloning. Cold Spring Harbor Laboratory
Press.).
For sequencing, the bacteria with the recombinant
plasmids were grown, and the plasmid DNA was puri-
fied (Sambrook et al. (1989). Molecular Cloning.
Le A 28 649 - 61 -
Cold Spring Harbor Laboratory Press.). Subsequently
the DNA was cleaved with PstI, and the cleavage
products were again separated by electrophoresis in
a 0.6~ agarase gel and electroeluted (Sambrook et
al. (1989). Molecular Cloning. Cold Spring Harbor
Laboratory Press). The isolated DNA fragments were
inserted into the sequencing vector pEMBL 19 and
again replicated in E.coli C600 (Sambrook et al.
(1989). Molecular Cloning. Cold Spring Harbor
Laboratory Press.).
To identify the gIV gene in the strain N569, the
genomic DNA thereof was cleaved with PstI, and the
fragments were separated by electrophoresis in a.
0.6~ agarose gel in TA buffer. The DNA fragments
were subsequently transferred to a nitrocellulose
membrane by Southern blotting (Sambrook et al.
(1989). Molecular Cloning. Cold Spring Harbor
Laboratory Press.) and hybridised with the radio-
actively labelled HindIII L fragment of the SH
strain (Sambrook et al. (1989). Molecular Cloning.
Cold Spring Harbor Laboratory Press.). A fragment
10.1 kbp in size was identified by means of the
hybridisation reaction as carrier of the gIV gene in
N569. After identification of the gIV gene, the
corresponding 10.1 kbp fragment was isolated by gel
electrophoresis and electroelution and inserted into
the PstI cleavage site of the vector pUCl2 using T4
ligase in analogy to the procedure for the SB and
Ausl2 strains. For this purpose, tha DNA of the
Le A 28 649 - 62
vector was treated with Pstl, recircularisation was
prevented by elimination of the 5'-phosphate group
using alkaline phosphatase, and the linearised DNA
of the vector pUCl2 was incubated with the isolated
10.1 kbp fragment and T4 ligase.
6.2 Sequencing
The sequencing of the Pstl fragments was carried out
by a method which is also called "chromosome walk-
ing". It is very suitable for the rapid sequencing
of large inserts from an existent clone, without
further subcloning. This entailed employing as
primers synthetic oligonucleotides which hybridised
at different points on the insert and starting the
Sanger sequencing reaction there. For this, the
fragments were sequenced starting from the flank of
the Pstl cleavage site. Towards the end of this
sequence a hybridising oligonucleotide was then
prepared and used in place of the universal primer.
This new primer was then used for further sequencing
into the i:ragments, and then the abovementioned
procedure was repeated. Since the resulting
sequences did not overlap in some regions, the frag-
ments were additionally specifically truncated with
the ExoITIfSI system, ligated and cloned in E.coli
C600. Thee~e subclones were sequenced and the
sequences yielded the required overlaps with the
sequences already present.
Le A 28 549 -
zo~~~.~~
7. Determination of serum-neutralising antibodies
against BHV-1
The serum neutralisation test for determining
neutralising BIiV-1 antibodies was carried out in
accordance with the instructions in Virologischen
Arbeitsmethoden volume II (Mayr et al. (1974).
Gustave Fischer Verlag, Stuttgart).
8. Coupling of the Peptide to BSA as carrier protein
8.1 Pep2 "
The synthetically prepared Pep2 has the amino-acid
sequence of the peptide segment AA317 to AA333 of~
gIV of the BHV-1 Schonboken strain:
gly-gly-ala-glu-gly-pro-lys-pro-gly-pro-ser-pro-asp-
ala.
8.2 Pep3
Pep3, which was likewise chemically synthesised,
has, besides the 15 amino acids of Pep2, another 15
amino acids of the Schonb8ken gIV. Tt comprises the
sequence of the peptide segment from AA307 to AA338
in gIV of the BHV-1 SB strain;
asp-gly-glu-ser-gln-thr-pro-glu-ala-asn-gly-gly-ala-
glu-gly-glu-pro-lys-pro-gly-pro-ser-pro-asp-ala-asp-
arg-pro-glu-gly.
he A 28 649 - 64 -
2085101
8.3 Coupling of snythetic peptide to BSA
5_ mg of synthetically prepared peptide (Pep2 or
Pep3) were dissolved in 1 ml of PBS pH=7.4 and
adjusted to pH 7Ø 11 mg of BSA in the case of
Pep2, and 6.2 mg of BSA in the case of Pep3, were
dissolved in 1 ml of PB5 and added to the vigorously
stirred peptide solution. Subsequently 2 ml of a
0.2% glutaraldehyde solution in PBS were added
dropwise to the continuously stirred peptide-BSA
mixture. The complete mixture was incubated at room
temperature overnight and, the next day, 0.8 ml of
a 1M glycine solution in PBS was added by pipette,
stirred for 1 hour, and then the conjugate wasw
dialysed against PBS (pH=7.4) for 5 hours, divided
into portions and frozen at -20°C.
9. ELISA for BHV-1 antibody determination (CV ELISA) in
bovine sera
The antigen employed in the complete virus ELTSA was
gradient-purified virus particles of the BHV-1 SH
strain. The Tmmulon 96-well plate (F form, Dynateeh)
was coated with 50 ng of antigen in 50 ~1 of coating
buffer ( 1. 06 g of NazC03, 2. 93 g of NaHC03 ad 1. O1
distilled water, pHm9.6) per well. The plates were
then incubated at +37°C in a COZ incubator for 16
hours. 3 Washes with washing buffer (PBS + 0.05$
Tween 20 + 1M NaCI) were followed by charging the
plates, apart from the blank, with blocking buffer
Le A 28 649 - 65 -
CA 02085191 2002-07-22
23139-7445
{200 ~1 PHS/Tweeri + 1M NaCl + 2% ovalbumin). Hoth
the plates and the test sera diluted 1:100 in
- blocking buffer were incubated at 37C for 1 hour.
3 Washes With washing buffer followed by 100 ~1 of
serum being pipetted per well (diluted 1:100 to
1:51200 in 2-fold series) in the plate for duplicate
determinations, and the plates were subsequently
incubated at +37C in a humidity chamber for 2
hours. The plates were thoroughly washed and then
charged with 50 ~l of POD-coupled rabbit anti-bovine
conjugate per well (diluted 1:2000 in PBS + 0.05%
Tween 20). The plates were incubated again at +37C
for 2 hours, washed, charged with 100 ~1 substrate
solution (11 mg of ARTS dissolved in 2.1 g citric
acid to 100 ml of distilled water, p8 ~ 4.2) and
incubated in the dark for 60'. The optical density
was measured at 405 nm. The serum dilution which led
in the ELISA to an extinction of 0.1 was defined as
antibody titre. Sera with a titre > 1:1000 were
assessed as BHV-1 positive.
The following controls were made up for each ELISA:
Control Antigen 1st Ab 2nd Ab Substrate
Blank no no no yes
Antigen control yes no yes yes
Antibody control no yes yes yes
Conjugate control no no yes yes
- 66 -
CA 02085191 2002-07-22
23189-7445
10. ELISA with synthetic peptide (Pep2) as antigen for
bovine sera to quantify antibodies against gD of
_ HHV-1 (Pep ElISA)
The Immulori"' 96-well plate in the F form, supplied by
Dynatech, was employed in the Pep ELISA. Pep2
coupled to BSA (see point 6) 100 ng/well, in 50 ~1
of coating buffer, was employed as antigen.
The coated plates were incubated at +37C in a
humidity chamber for 16 hours. 3 Washes with washing
buffer (PBS + 0.05% Tween 20) were followed by all
wells being charged with 200 ~1 of blocking buffer
(PBS + 0.05% Tween 20 + 1% skimmed milk powder, orw
the test sera being diluted 1:100 in blocking buffer
+ 1.5M NaCl. Both the blocked plates and the diluted
sera were incubated at +37C for 1 hour. Then 200 ~1
of each of the sera to be tested, diluted 1:100 to
1:3200 in blocking buffer + 1.5M NaCl, were pipetted
into the plates, and the plates were incubated at
+37C for 2 hours. Subsequently the plates were
washed 3 x with washing buffer, and 50 ~1 of rabbit
anti-bovine-POD conjugate (diluted 1:2000 in PBS +
0.05% Tween 20) were added to each well, incubated
at +37C for 2 hours, the plates were again Washed
3 x with washing buffer, and 100 ~1 of substrate
solution (11 mg of ABTS dissolved in 98 ml of
substrate buffer (2.1 g of citric acid ad 98 ml with
distilled water, pH=4.2)) were pipetted into each
well. The colour reaction was stopped with 100 ~1 of
- 67 _
CA 02085191 2002-07-22
23189-7445
1% strength SDS solution after incubation at +37°C
in the dark for one hour. The measurement was
w carried out at 405 nm.
The following controls were made up for each ELISA:
Control Antigen 1st Ab 2nd Ab Substrate
Blank no no no yes
Antigen control yes no yes yes
Antibody control no yes yes yes
Conjugate control no no yes yes
Evaluation:
Sera for which the extinction of the 1:100 dilution
less the extinction of the antibody control was
z 0.100 O.D were assessed as HHV-1 positive.
Sera for which the extinction of the 1:100 dilution
less the antibody control was < 0.100 O.D were
assessed as BeV-1 negative.
11. Dot-blot
Before protein loading, the carrier membrane
(ImmobilieriMP, Millipore) was briefly wetted with
methanol and subsequently washed thoroughly with
distilled water and stored in PBS (pH 7.4) until
used. 0.5 ~g and 1.0 ug of synthetic peptide (Pep3
- 68 -
208~~.9.~
coupled to BSA), or 1.0 ~cg of BSA each in 100 ~1 PBS
were loaded on to the membrane filter.
The free binding sites on the Immobilien membrane
were subsequently blocked with blocking buffer (PBS
+ 0.5% skimmed milk powder + 0.05% Tween) for 2
hours. The membrane was then incubated with the
first antibody (diluted 1:50 in PBS + 0.05% Tween)
for 2 hours, washed 3 x (PBS + 0.05% Tween), and
incubated with the 2nd antibody (anti-bovine-POD
conjugate; Sigma) (diluted 1:2000 in PgS + 0.05%
Tween) for a further 2 hours and again washed 3 x in
PBS + 0.05% Tween. All the incubation steps were
carried out at room temperature. After the last--
washing, freshly made up substrate mixture:
- 20 mM NaCH3C00 solution, pH 5.0-5.5
- 10 mg of 3-amino-9-ethylcarbazole dissolved in
3 ml of DMSO
- 0.04 ml of a ~0% strength HZOZ solution
was added and the coloration was evaluated after 15
minutes. Binding of Pep3-specific antibodies to Pep3
was thus signalled by a colour reaction on the
membrane.
12. Bovine sera
Bovine sera from animals which had been infected
with the following BHV-1 strains were available for
Le A 28 649 - 69 -
the serological investigations in the Pep ELISA,
CV ELISA, SNT and dot-blot:
- Australial2
- N569
- Sch~nbciken
- Schleswig-Holstein
In addition, field sera frown Northeraa ~eranany with
a known content of BHV-1 specific antibodies, tested
in the ELISA, were employed in Pep ELISA, CV ELISA
and SNT.
II Results
1. Heterogeneity of size in gIV
In the following investigations, the gIV frown BHV-1
SB, Ausl2 and N569 strains, prepared as described
under point 4, was investigated far its size in gel
electrophoresis. The molecular weights of the
proteins were deduced frown their migration behaviour
in the gel.
The gIV in the samples investigated by gel electro-
phoresis showed distinct differences in size in the
Ausl2, SB and N569 strains. The size of gIV was 84KD
in the case of Ausl2, 72K1D for SB and 68KD for N569.
A heterogeneity of size can in theory be caused by
Le A 28 649 - 70 -
2Q~5~9~
open reading frames (ORF) of different size in the
gIV gene of the various BHV-1 strains. Other possi-
bilities are different co- and post-translational
modifications of gIV.
To characterise the ORF of gIV in various BHV-1
strains, the complete sequence of the gIV gene was
determined for the Ausl2, N569 and SB strains,
compared with one another, and the amino-acid
sequence of the protein was deduced therefrom.
Fig. 1 shows for comparison the amino-acid sequence
of gIV for the Ausl2 and SB virus strains, Fig. 2
for the N569 and SB strains.
In the case of Ausl2, the ORF of gIV comprises
1524 by which code for 507 amino acids, in the case
of SB 1254 by which code for 417 amino acids. A
repetetive sequence is responsible for the hetero-
geneity in size of the ORF in the gIV genes. This
repeat sequence 90 by in size is present as direct
repetition in the Ausl2 strain in a copy number of
4, in the SB strain in a copy number of 1. In the SB
strain, this repeat sequence is located from by 1022
to by 1111 of the ORF and codes for amino acids
306-335 with the following sequence:
P-E-G-D-G-E-S-Q-T-P-E-A-N-G-G-A-E-G-E-P-IC-P-G-P-S-
P-D-A-D-R.
In the case of Ausl2, this repeat sequence extends
Le A 28 649 - 71 -
2085191
from by 1318 to by 1408 of the ORF and codes for
amino acids 306-425, a 4-fold copy of the sequence
" is the SB strain.
The N569 strain has a different amino-acid sequence
than the SH and Ausl2 strains in the region of the
repeat sequence, which is present only in one copy
in it, exactly as in SB.
In this region there are only 19% homology between
the two strains, SB and N569, investigated, compared
with 81.5% homology based on the complete gIV
protein in these two BHV-1 strains. The sequence in
the case of N569 in the region of the repeat reads:
E-G-P-A-A-A-G-P-D-G-P-P-P-G-E-P-R-P-G-P-G-G-P-G-A-
D-V-D-R.
Since the gIV both in SB and in N569 comprises 417
amino acids it is possible that the heterogeneity in
size observed in samples, investigated by electro-
phoresis, in gIV of these two strains is not based
on ORFs of different size. On the contrary, in the
N569 strain there is substitution of the amino acid
309 in position 44 by the amino acid isoleucine. The
loss of a glycosilation Bite which is associated
with this is presumably the reason for the hetero
geneity in size, observed in the gel
electrophoresis, of gIV in SB and N569.
Ls A 28 649 - 72 -
2~~~191
2. Computer analysis
The repeat region was identified as potential
antigen (hydrophilic region) by means of the
Chou-Forman prediction for the gIV protein of the SB
strain.
3. Antigenicity in cattle
Bovine sera taken before and after infection with
the BHV-1 Ausl2, SB, SH and N569 strains, or field
sera from cattle with known BH'V-1 antibody content,
were investigated in the Pep ELISA for Pep2-specific
antibodies, in the CV ELISA and SNT for BHV-1--.
specific antibodies and in the dot-blot for Pep3-
specific antibodies.
3.1 Australia 12 sera
Tab. 1 shows for comparison the results of 18 bovine
sera Pram 7 animals before and after infection with
the BBV-1 Ausl2 strain. For the characterisation,
the sera were investigated in the Pep ELISA, CV
ELISA, SNT and dot-blot for Pep- and BHV-I-specific
antibodies. No antibodies directed against B13V-1
virus particles, Pep2 and Pep3 were detectable in
any of the lbovine sera before loading infection in
the SNT, CV ElISA, Pep ELISA and dot-blot.
It was possible with the SNT to detect in all sera
Le A 28 649
after infection, except in the serum of OM 25123
after first infection and in the serum of OM 25126
- after booster infection, virus-neutralising BHV-1
antibodies. In the case of OM 25123, neutralising
BHV-1 antibodies were detectable only after booster
infection.
It was likewise possible with the CV ELISA to detect
BgiV-1-specific antibodies in the same sera after
infection in which virus-neutralising antibodies
were detectable, with the exception of the serum of
OM 25124 after First infection.
Antibodies directed against Pep2 were likewise
detectable in the ELISA fox sera which showed serum-
neutralising antibodies. An exception was the serum
of OM 87 after infection, which indeed had virus-
neutralising BBV-1 antibodies in the SNT but had no
Pep2 antibodies in the ELISA.
No BBV-1 and Pep2-specific antibodies were detect-
able in the serum of the bovine OM 25123 after the
first infecwtion and in the serum of the bovine
OM 25126 after reinfection, both in the serum
neutralisation teat and in the Pep ELISA.
Antibodies directed against Pep3 were detectable
with the aid of the dot-blot in sera which were
taken after the first infection with BBV-1 and which
had virus-neutralising antibodies. In addition, it
Le A 28 649 ~ 74 -
was possible to detect with the dot-blot antibodies
directed against Pep3 in the serum of 0M 25123 which
was taken after infection (on 21.09.90) and which
contained no serum-neutralising and ELISA anti-
s bodies.
3.2 N569 sera
Tab.2 shows, in analogy to Tab.l, for comparison the
results of bovine sera before and after infection
with the HHV-1 N569 strain. For the characterisa-
tion, the sera were investigated for Pep2- and B6IV-
virus particle specific antibodies in the Pep ELISA,
CV ELISA and SNT. _-
Before infection with the BFiV-1 N569 strain, no
antibodies directed against B13V-1 or Pep2 were
detectable in any of the bovine sera both in the
SNT, CV ELISA and in the Peg ELISA. Following the
infection, BBV-1-specific antibodies were detectable
in both sera in the SNT and CV ELISA. Antibodies
directed against Pep2 were, however, not to be found
in the Pep :ELISA even after loading infection.
Thus, although the cattle infected with N569 pro-
duced BHV-1-specific antibodies which react in the
CV ELISA and SNT, did not recognise as antigen in
the Pep ELISA after infection the Pep2 which was
detected in the gIV of the SB and Ausl2 strains but
not identified in the case of N569.
Lc A 2a 649 ~ - 75
~o~~~~~
3.3 Schleswig-Holstein and Schonb~ken sera
- Tab. 3 shows for comparison the results of bovine
sera before and after infection with the BHV-1 SB
and SH strains. These sera were also investigated
for Pep- and BHV-I-specific antibodies in the Pep
ELISA, CV ELISA, SNT and dot-blot for the character-
isation. Antibodies directed against BHV-1, Pep2 and
Pep3 were detectable in no tested serum before
infection with the BHV-1 SH and SB strains, and in
all bovine sera after infection, in the SNT, CV
ELISA and Pep ELISA.
3.4 Field sera
Tab. 4 shows the results of the investigations of
bovine aera from controlled stocks whose BHV-1
antibody status had been tested in the ELISA. The
sera were investigated for BHV-1 and Pep2-specific
antibodies in the Pep2 ELISA and CV ELISA.
BHV-1-specific antibodies were detectable in 29 of
the 31 tested positive sera with the CV ELISA.
Antibodies directed against Pep2 were found in all
31 positive sera in the Pep ELISA.
No BHV-1- a;nd Pep2-specific antibodies were detect
able in the negative sera in the case of the 6 sera
tested in the CV ELISA and in the case of 5 of the
6 sera tested in the Pep ELISA.
Le A 28 649 - 7g -
~~8~~~1
4. Immunogenicity testing of the N569 strain
Tab. 5 shows the results of the investigations of
bovine sera in the SNT after immunisation with the
N569 strain. The 4 cattle were immunised with N569
strain in different ways.
Serum-neutralising antibodies were detectable in
serum from the immunised animals no later than 13
days after immunisation. The highest antibody titre
was reached by the bovine immunised intravenously
(i.v,), with a titre of 1:152. Antibody levels of
1:45 and 1:27 were reached after intramuscular
(i.m.) and intranasal (i.n.) administration of the
N569 strain, and the antibody level reached after
intravaginal (i.vag.) administration was 1:9.
5. Conclusion
It was possible by combined comparison, of the
antibodies directed against the modified amino-acid
sequence of the N569 strain of antibodies directed
against BHV-1 virus particles, to differentiate
between cattle free of BHV-1 antibodies, animals
immunised with the N569 strain, and cattle infected
with field strains.
The BHV-1 N569 strain proved to be immunogenic for
cattle after i.v., i.m., i.n. and i.vag.
Le A 28 649 - 77 -
administration.
Description of the figures~
Fig. 1 shows for comparison the amino-acid sequences of
the gIV of the BHV-1 Ausl2 and SchonbSken strains. The
amino-acid sequence and homologies were identified by the
PC program PC-Gen. The upper sequence belongs to the
Ausl2 strain, and the lower sequence to the Sch~nboken
strain. The sequences start at the 5' end (N terminus) of
gIV and end at the 3' end (C terminus). Lines between the
two strands indicate homologies. The arrows drawn in
identify the amino acid cysteine.
Fig. 2 shows for comparison the amino-acid sequences of
the gIV of the BfiV-1 N569 and Schonboken strains. The
amino-acid sequence and homologies were identified by the
PC program PC-Gen. The upper sequence belongs to the N569
strain, and the lower sequence to the Schonboken strain.
The sequences start at the 5' end (N terminus) of gIV and
end at the 3' end (C terminus). Lines between the two
strands indicate homologies. The sequence of the synthe-
sired Pep3 is underlined. The box in position AA41-43
identifies the absent glycosilation site of the BHV-1
N569 strain.
Tab. 1 shows fox' comparison the results of bovine sera
before and after infection with the BHV-1 Ausl2 strain.
The sera were investigated for Pep- and BI3V-1-specific
antibodies in the Pep ELISA, CV ELISA, SNT and dot-blot
Le A 28 649 - 7g _
Image
___ _~._________.___~._~._
c7.'~0 ~~~~~ a ~~
a
N N~N~NQ'~N~~N ydlv7 r
r~d f°~ ~ ~ 'p~
4
-.,
_ _ ..e .- ~.. _ _ e. . . o _ _ ~ _ . _ ~.
~ a _-.. _ _. .~ _ _ .- _ _ _ _ .- _ _
u9 N eCf
__
~°~'~ o~°~o~$~~~~~~~~~~~
cwcQoo~o~oooooocas'o~
U' ~~~~~~~~~a~~~~~~~~~
A~~~oo~~~
o .~ o
~o~ a a
..- ~~':~~'.~'H,NH vN~i~~~..1~'d'~~.N.~'.~~vNH
H Y f.i H Ii 4 t~. 1r y y v ~ ~ d ~ ~ a~d
___ ____u ~_____~______
a
, ~ c ~ ~ ,.., o°v ~
~~4 -r~ y .N ~.i ~ .r; .,~ .re ..~ ..r r
-.a..w ~ ~ ~,r ~.a ~ II
_ U U _ _ ~ _
_ . _ _ _ . ..
b~s~~~ ~ $..~r ~~ w~ $~ ~~ ~ aJ
a II a a
U
8 ~ '08 0'8 ~ ~ g~ !Q ~3 ~3'~4 ~ LE3 ~4 '~3 !4'A ~ ° ° v
~C
.,.~ .,./ .,./ .~
Le A 28 649 - 8p
Tab. 2 shows for comparison the results of bovine sera
before and after infection with the BHV-1 N569 strain.
The, sera were investigated for Pep- and- BHV-1-specific
antibodies in the Pep ELISA, CV ELISA and SNT for the
characterisation.
Le A 28 649 - 81 -
zo~~.~~~
- N -
-- EN
N
v v~
W -
1
H
N
- --_a
E
- ----
0
~r1 _
V N
1 P1
-l
p
A
1 OOOCi
c~ooo
~
o ,
o
c
o
o, o,
sn
o~~
i ~~
-'~
.~i
~i.ro
~ s~sa
N
Le A 28 649 _ 82 _
Tab. 3 shows for comparison the results of bovine sera
before and after infection with the BHV-1 S19 and SH
strains. The sera were investigated for Pep- and HHV-1
specific antibodies in the Pep ELISA, ACV EI,~SA, SNT and
S dot-blot for the characterisation.
Le A 28 X49 - 83 -
~~~~~9.~
-__ ~ _____.~_______
_-_ ~ __--~.-___~~~__
N Pi N ~ N G N ~ L1
D C B
-. _ _.~. _ _ _ _ _ _ _ _ _
~0 b 'A P ~~.1
"" ~ ~ ~~ B B G ~
~ °~ 'B 'A R9 'd '~ '~9 °~
C G B C O f, B C.
.._ _ _ __ _ _ _ _ _ _ _ _ _
O
~7 N ~ ~ ~ ~ ro 't7 'A '~ '~ '~ 'G b b
d ~ ~ i C Q 04 ~ C a G A C C C 4
_ _____ __..~..a____
~ O NN
t~ e0 s0 b "D 'v "~ '~ b b 'r! 'O i
~ . ~ s ~~
~~4~~=CCC6~'Cf:
o ~ Q c a a a a c ~i a a
w b w b w °a w ~ ~e9
a a ~ c c a c ~ ~
-.- ~~~i3~a3~~~~~~3~~
w .~ .~ orer yr O .-1
e w w a~
,ar v sr
a nr
r1
~,.1
r
~.l
w1
r1
wi
w~l
wH
~t
r1
~ni
~.1
O
__ ~
N N
H
__ ~t~N~Vi~ IN
~~
~~
t~s~~f~
*
L~ A 26 649 - ~4 -
Tab. 4 shows the results of pep2 ELISA, CV ELISA and SNT
with field sera of known HHV-1 status. 31 positive sera
and 6_negative sera whose BHV-1 antibodies status had
been determined by ELISA by the Oldenburg Animal Health
Service were available for the investigations. All the
sera were characterised in the pep ELISA and CV ELISA. 3
positive sera were tested in the SNT.
Le A 28 649 - 85 -
20~~~.~.~
Tab. 4
1 Pep I ~ I SNT
I F~,L.ISA ~~ I
Cd.D.405
~n~
I HvA Pep2 Pep I CV I Titre
I Pep2 I
I O.De Ab I I El..~.sa/~1 ;[ . 1~~~1
p.xua C Ab ' ~~
1 C
Nu pas.1 0,293 0,000I 1 per.1 37691 pas. ( n.d.
F~* 0,293 1
I
I~t2 ( 0,518 0,049( 1 poe.I 11487I per. 1 n.d.
pos. FS* 0,469 I
1
Id~f3 1 0,330 0,0(70I I pos.I 24681 p~s I 384
poe. FS* 0,330 I
I
PM4 I 0,364 0,117I ( pos.I 3b34( pas. 8 n.d.
pas. hS* 0,247 I
I
Ar96 I 0,520 0,228I 1 poe.I 2399I pos. I n.d.
pos. FS* 0,292 I
I
N~fl I 0,458 0.1521 I ~. I 2480I pas. 1 n.d.
pas. F'S* 0,316 I
1
I~ pas.I 0,537 0,218I I pas.I 17~ I pas. 1 n.d.
F'S* 0,319 I
I
td~ I 0.219 0,004I I pas.I 6583I pas. I n.d.
per. E:S* 0,215 1
I
lJ~ilO 1 0,366 0,215I ( pos.i 5760I pas. I n.d.
p08. FS* 0,151 .I
I
I~fill I 0,272 0,169I I pos.I 14821 pos. I n.d.
pos. FS* 0,103 I
I
MI12 I 0,251 0,113I I pos.I 5404i pos. I n.d.
pos. F'S* 0,138 I
I
f~tl3 1 0,522 0,1411 I pos.I 2677I pas. I r~.d.
pos. FS* 0,381 I
I
Mdl4 I 0.361 0,165I 1 per.I 88?3I pos. I n.d.
poa. FS* 0,196 1
I
PM15 I 0,395 0,225I I pos.I 13b3I pos. I 448
pas. F5* 0,170 1
1
i~116 I 0,353 0,115I I pos.I 1376I Pos. I 224
pos. F5* 0,238 I
I
1017 I 0,351 0,111I I pas.I 3b8 I toeg.I n.d.
pos. F:s* 0,240 .1
I
N418 I 0,3~ 0,1371 1 pays.I 1511I pes. I n.d.
mss. FS* 0,183 I
I
IdRl9 I 0,359 0,172I I p08.I 947 I neg. I n.d.
pas. FS* 0,187 I
I
t4~0 i 0,425 0,226I I pos.I 1060I poe. I n.d.
pos. FS* 0,199 1
I
M121 I 0,576 0.135I 1 poe.I 1842I poe. 1 n.d.
pas. FS* 0,441 I
I
1122 I 0,599 0,1511 I poe.I 1264I pos. I n.d.
pos. FS* 0,448 I
I
1123 I 0,474 0,118I I pae.I 1391I pas. 1 n.d.
pae. FS* 0,356 I
1
NP129 I 0,582 0,118I 1 pos.I 2133I poa. I n.d.
poa. FS* 0,464 I
1
I~i25 I 0.348 0,092I I ~. I 1805I pas. 1 n.d.
pas. F8* 0,256 I
I
I~126 I 0,264 0,129I I pos.I 1295I pae. 1 n.d.
pos. FS* 0,135 I
I
N~fL7 1 0,325 0,086I I per.I 14811 pos. I n.d.
pae. F'S* 0,239 I
I
NM28 I 0,348 0,154I 1 pos.I 2355I pos. I n.d.
pas. FS* 0,194 I
1
N~29 1 0,368 0,1171 I poa.I 2967( pos. ( n.d.
pog. FS* 0,251 I
I
t~C~O 1 0,261 0,1361 I poa.I 6568I pas. I n.d.
pas. FS* 0,125 I
I
Mt31 I 0.493 0,152I I pos.I 4416I pos. I n.d.
pos. FS* 0,341 I
I
Neil I 0,092 0,008I I aeeg.I 310 I r~e9.1 n.d.
neg. FS* 0,084 I
(
id~l2 I 0,097 0,0041 1 neg.I 390 I n~eg.I n.d.
neg. F5* 0,093 I
I
N13 I 0,212 0,007I I poe.1 281 I neg. I n.d.
peg. FS* 0,2D5 I
(
M!4 I 0,204 0.105I I neg.1 748 I neg. 1 n.d.
neg. FS* 0.099 I
I
DDS I 0,096 0,048I I neg.I 560 I rs~g I n.d.
neg. E'S* 0,048 i
I
t~16 I 0,030 0,043I 1 neg.( 562 1 n~. 1 n.d.
nag. FS* -0,013 J
I
* P'S ~ field sera with )gym 81:1<J-1 status, tested iaa the ELISA.
Le A 28 649 _ 86 _
Tab. 5 shows the reciprocal antibody titres of bovine
sera in the SNT after immunisation with the N569 strain.
Tab. 5
I Mode of immunisation
$
I L a ~ l . n Z a V ~ s V
a I . ( a ~ ~ o I
I
nets I oM e1 oM 83 oM 9o oM e9
__ i $ I I
____ __
_ _
11.07 j ' _C2____; _______ ~___'2____i
~2
12.07 I <2 I <2 I <2 $ <2 I
13.07 $ <2 I <2 I c2 I e2 I
14.07 I 2 I <2 I <2 1 <2 $
15.07 I <2 I <2 1 <2 I <2 I
16.07 $ <2 I t2 I <2 I <2 $
17.07 I <2 I <2 I <2 I <2 $
18.07 $ 3 I <2 $ e2 $ <2 I
19.07 I 2 I <2 I <2 1 3 $
20.07 I 2 $ <2 $ <2 I 6 i
21.07 I 4 $ 2 I <2 I 23 I
22.07 I 3 $ 2 I <2 I 45 $
23.07 I 4 1 6 I 2 I 90 I
24.07 I 5 I 11 I 2 $ 128 $
25.07 I 8 I 16 I 3 I 64 $
27.07 $ 3 I 13 I <2 $ 152
30.07 $ 9 I 16 I 7 I 152 I
01.08 $ 11 1 11 I 6 I 128 $
03.08 I 13 $ 27 $ 6 1 181 $
06.08 $ 27 $ 11 I 8 I 107 I
08.08 I 45 I 27 I 9 $ 152 I
_________$ _________.I_________I_________I_________I
Le A 28 649 - 87
