Note: Descriptions are shown in the official language in which they were submitted.
CA 02297507 2000-O1-31
SYNTHETIC GENE OF BOVINE VIRAL DIARRHOEA VIRUS
The present invention refers to synthetic Bovine Viral Diarrhoea virus genes,
live attenuated Bovine
Herpesvirus recombinants, live attenuated and inactivated Bovine Herpesvirus
recombinants
carrying such genes, vaccines based on these live attenuated recombinants,
methods for the
preparation of such live attenuated recombinants and to methods for the
preparation of such
vaccines.
Bovine Viral Diarrhoea virus (BVDV), a member of the Flaviviridiae, is a cause
of congenital and
enteric disease with a wide range of clinical manifestations. The disease was
first described by P.
Olafson et al. (Cornell Vet. 36 (1946) 205-213) as a transmissible disease of
cattle with a high
morbidity and low mortality. Affected cattle displayed elevated temperature,
diarrhoea, and
coughing. The condition was termed bovine viral diarrhoea.
Today, BVDV is considered to be a major pathogen of cattle with a world-wide
economic impact
(M.L. Doherty ML, Irish Veterinary Journal 40 (1986) 189-190; H.R. Frey et
al., Dtsch. Tierarztl.
Wschr. 94 (1987) 580-583; C.E. Ross et al., J. Am. Vet. Med. Assoc. 188 (1986)
618-619). The
clinical pattern of BVDV infection within a herd and consequently its impact
on production depend
on the interaction of several factors, including the strain of the virus, age
of the cattle, immunity in
the herd, and interplay of stressors.
BVD is an acute postnatal infection in seronegative immunocompetent cattle.
Acute BVDV infection
enhances clinical diseases caused by other pathogens and is an important
component of the
bovine respiratory disease complex. Postnatal exposure of immunocompetent
cattle to
concytopathic biotypes of BVDV causes clinical BVD of varying severity.
Naturally acquired BVD is
usually characterised by high morbidity, low mortality, a normal host immune
response, and
minimal mucosal lesions. Pyrexia, nasal discharge, and transient leukopenia
are frequently seen.
The failure to respond to BVDV infection until around day 125 of gestation can
result in embryonic
death, resorption, and stillbirths or, more frequently, in a whole array of
non fatal teratogenic effects
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(H.-J. Thiel et al., Fields Virology, Eds. B.N. Fields, D.M. Knipe, P.M.
Howley et al., 3rd Edn., Raven
Publishers Philadelphia 1996, p. 1059 ff.).
Since BVDV-infection leads to high economic losses, there is a substantial
need for vaccines
against BVDV.
To control the disease, inactivated vaccines are currently preferred. They
induce, however, only a
limited immune response which has been shown to be insufficient for protection
against antigenic
variants of BVDV (Bolin et al., Am. J. Vet. Res. 52 (1991 ) 1033-1037). Thus
there is a clear need
for improved, efficacious vaccines against BVDV.
However, vaccine development has been hampered because it is not known how a
protective
immune response can be induced by subunit or vector vaccines. First attempts
to vaccinate calves
using a baculovirus expressed glycoprotein E2 provided only limited protection
(Bolin and Ridpath,
Arch. Virol. 141 (1996) 1463-1477).
Since, as mentioned above, it is not known how a protective immune response
can be induced, it is
important for potential vaccines to at least mimic the natural infection as
closely as possible.
Therefore, in order to improve the immune response it is tempting to express
the immunologically
relevant BVDV E2 glycoprotein by a live recombinant vector virus.
These vector viruses mimic the natural infection in the sense that they do
infect host cells and
express, next to their own genetic material, the additional genetic
information cloned into their
genome.
An obvious vector for essaying the possible expression of the BVDV E2
glycoprotein under
laboratory conditions is the vaccinia virus. This virus has successfully been
used as an expression
vector for a multitude of different genes for many years (see e.g. Paoletti,
US 4,603,112 regarding
the general principle for integration of foreign DNA into vaccinia virus to
produce a modified virus
capable of expressing foreign genes. For a general review about vaccinia virus
vectors, see e.g.
D.J. Jolly, Semin-Immunol. 2(5) (1990) 329-339).
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For experimental purposes, the use of vaccinia virus as a live recombinant
vector virus is attractive,
because much is known about the virus and many tools for making vaccinia
recombinants are
available.
Vaccinia virus, however, is known to have an extremely broad host range. It is
known to be
infectious for all mammalian species tested so far, ranging from rabbits,
mice, racoons, sheep,
goats and camels to humans (D.J. Jolly, Semin-Immunol. 2(5) (1990) 329-339).
From an environmental point of view, this broad host range of live recombinant
viruses in general is
an unwanted situation, especially where only bovine animals are target animals
for BVDV-
vaccination. Vaccinia virus, although a bovine pathogen, would therefore
certainly not be the vector
of preference for use in animal vaccines for the protection of bovine animals
against BVDV.
Promising live recombinant vector virus candidates however would be the live
attenuated bovine
herpesviruses (BHV), since such bovine herpesviruses, if expressing the BVDV
E2-gene, would
have the following advantages:
~ they are host-specific: they only infect bovine species;
~ they protect against two different diseases: Bovine herpesvirus infection
and BVDV-infection.
Live attenuated re~.ombinant BHV carrier viruses are known to be good
potential expression vectors
and vaccine viruses at the same time (R.S. Schrijver et al., Vaccine 15 (1997)
1908-1916; S.
Chowdhury, Vet. Microbiol. 52 (1996) 13-23; F. Fehler et al., J. Virol.66
(1992) 831-839; C.
Schroder et al., J. Virol. 70 (1996) 25-33; J. Schmitt et al., J. Virol. 70
(1996) 1091-1099; G.M. Keil
et al., J. Virol. 70 (1996) 3032-3038; E. Hanon, J. Virol. 70 (1996) 4116-
4120; J.C. Whitbeck et al.,
J. Virol. 70 (1996) 7878-7884; A. Miethke et al., J. Gen. Virol. 76 (1996)
1623-1635).
BHV-1 has e.g. been used for the expression of LacZ (J. Schmitt et al., J.
Virol. 70 (1996)
1091-1099). BHV-4 has been used e.g. for the expression of the LacZ-gene
(Vanderplasschen et
al., Virology 213 (1995) 328-340), and the herpes simples virus I thymidine
kinase gene (Keil, G.M.,
Vlllth International Congress of Virology, Berlin, 26-31 August, 1990).
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Until now, however, expression of BVDV genes cloned in BHV as a live
attenuated recombinant
carrier virus, let alone showing in vivo protection by such a BHV-recombinant,
has not been
reported in the art. This is due to the fact that BHV is
replicating/expressing in the nucleus, whereas
BVDV is replicating/expressing in the cytoplasm.
Two main problems are known to exist when genes of a cytoplasmatically
replicating virus are
expressed in the nucleus, which is an evident consequence of cloning into the
genome of a virus
that replicates in the nucleus, e.g. BHV:
a) For genes from cytoplasmatically replicating viruses, e.g. the E2 gene of
the closely related
classical swine fever virus - which is also a member of the Flaviviridiae - it
has been shown
that the presence of cryptic splice signals caused unwanted splicing of E2
mRNA in the
nucleus of mammalian cells. This is explained in more detail below.
Consequently no
expression of the E2 glycoprotein occurs (J.S. Shiu et al., J. Virol. Meth. 69
(1997) 223-
230). The problem of the existence of cryptic splice sites is a general
problem when
expressing genes originating from cytoplasmatically replicating viruses in the
nucleus.
b) Moreover, cytoplasmatically replicating viruses have a low G/C content. For
genes of such
viruses to be expressed successfully in the nucleus, the low G/C content of
the genes to be
expressed must necessarily be brought up to the high G/C level of the genes of
the vector
virus. This necessity was shown e.g. for the related Bovine Respiratory
Syncytial Virus
(BRSV; R.S. Schrijver et al., Vaccine 15 (1997) 1908-1916; G. Kiihnle et al.,
J. Virol. 72
(1998) 3804-3811 ). Expression of the G glycoprotein of BRSV by recombinant
BHV was
only possible after increasing the G+C content of the open reading frame (ORF)
from 42
to 62 % and by simultaneous removal of all potential splice donor signals. The
same has
been shown for the BRSV-F-gene.
Comparison of the GC-content of BVDV-genes and BHV-genes showed the following:
the average
GC-content of the BVDV-E2-gene is about 46 %, whereas the G/C content of e.g.
the BHV-gD-
gene is about 72 %. Therefore, on the basis of the prior art, it was to be
expected that the GC-
content of the synthetic BVDV-gene had to be raised dramatically by replacing
the nucleotides A
and T by G and C, as far as possible within the framework of what is allowed
by the degeneracy of
the genetic code. This is an arduous and labour-intensive task since first of
all for each G/C-
enhancing change in each codon, it must be checked if the changed codon still
encodes the native
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amino acid. Secondly, after this has been done, the whole G/C-enriched E2 gene
has to be
synthetically prepared.
Surprisingly it was found now, that if the original native BVDV E2-gene is
replaced by a synthetic
BVDV-gene having a synthetic sequence from which only splice-sites were
removed, and still
encoding the naturally occurring amino acid sequence, the gene is successfully
expressed in the
nucleus. This is fully unexpected in view of the extreme difference between
the BVDV GC-content
(46 %) and the average GC-content of BHV-genes, which is above 70 %.
Therefore, one embodiment of the invention relates to synthetic BVDV-genes
having a not naturally
occurring nucleotide sequence, from which only splice-sites were removed but
still encoding a
naturally occurring amino acid sequence.
Since the E2-protein is an important immunogenic protein of BVDV, a preferred
form of the
invention relates to the synthetic BVDV-gene encoding the BVDV E2 protein.
The use of BHV (replicating in the nucleus) for the expression of BVDV genes,
normally replicating
in the cytoplasm, may lead to the formation of unstable (BVDV-)RNA due to the
presence of
obvious and/or cryptic splice-donor and/or -acceptor sites, that are
recognised by the nuclear RNA-
splicing mechanism. Splice-sites have small consensus sequences for both
splice-donor sites
(GTRAGT as a consensus sequence where R is A or G; splice donor sites can also
be GTATGT
and GTAGAT) and splice acceptor sites (Y~NYAG where Y is C or T, and N can be
any nucleotide).
In e.g. the nucleotide sequence of the wild-type E2-gene at least four
potential splice-donor sites at
positions 152-157, 419-424, 614-619, and 1061-1066 matching the AGGU consensus
sequence
were found, which are not present in the synthetic E2-gene.
Consensus sequences of both the splice-donor- and -acceptor as known in the
art, have been
mentioned above. Splice-sites can be removed by replacing one or more
nucleotides of the
potential splice-site within the framework of what is allowed by the
degeneracy of the genetic code.
This can be done by e.g. site-directed mutagenesis, or replacement of small or
longer fragments by
fragments with an alternative sequence. Alternatively, the whole gene can be
replaced by synthetic
DNA-fragments. Techniques for site-directed mutagenesis, and for DNA-synthesis
are known in the
art.
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It may not be necessary to remove all potential splice sites, since in many
cases, the removal of
only a splice-donor, or splice-acceptor site will suffice to suppress
splicing. Therefore, a more
preferred form of this embodiment relates to the BVDV E2-gene modified in such
a way that at least
one possible splice-donor or acceptor site found in the naturally occurring
nucleic acid sequence is
removed in the synthetic BVDV-gene.
Comparison of the nucleotide sequence of the synthetic E2-gene according to
the invention and the
original E2-gene shows, that the GC-content of the synthetic gene is only
slightly higher than the
GC-content of the native E2-gene. Due to the removal of splice donor sequences
the G+C content
of a synthetic E2 ORF inherently increased from 46 % to approximately 48%.
Nevertheless the
synthetic gene of the present invention can, in spite of its very low G/C
content, surprisingly efficient
be expressed in BHV-recombinant viruses under the control of eukaryotic
promoters, and the so
obtained BHV/BVDV-recombinant viruses can be propagated in a stable manner.
Therefore, in an even more preferred form, the synthetic BVDV E2-gene
according to the invention
has a GC content between 46 and 60 %, or even between 48 and 50 %.
Although it is in principle possible to fully adapt the GC-content to the high
GC-content of BHV, a
very efficient expression was already obtained when the GC-content was 48%.
Thus in a still even
more preferred form, the synthetic BVDV-gene has a GC-content of approximately
48 %.
A naturally occurring nucleic acid sequence is understood to be a nucleotide
sequence as it is
found in naturally occurring viruses, such as the viruses isolated from the
field.
The synthetic gene according to the present invention has a nucleotide
sequence that, albeit
different from the original nucleotide sequence (i.e. the nucleotide sequence
found in virus isolated
from the field), still encodes exactly the same amino acid sequence, and thus
encodes the native
protein. This is possible due to the degeneracy of the genetic code: all amino
acids (with the
exception of Met and Trp) are coded for by at least two different triplets. In
the case of Leu, there
are even 6 different triplets encoding this amino acid. Thus, there is a large
number of possibilities
to modify the nucleic acid sequence encoding a certain gene, without at the
same time modifying
the amino acid sequence. This principle is applicable to each and every gene
and therefore also to
the BVDV E2-gene.
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The degeneracy of the genetic code also allowed e.g. the introduction of a
number of restriction
enzyme cleavage sites in the nucleic acid sequence encoding the E2-protein
gene, without
modifying the amino acid sequence of the E2-protein.
As an example may serve the modification of the BVDV E2-gene made from
position 599 onwards,
where the sequence GGCTCT was replaced by GGATCC, thus providing the BamHl
restriction site
at position 600 without changing the amino acids glycine and serine encoded by
these two triplets.
New unique restriction enzyme cleavage sites for Pstl, Hindlll, Eagl, BamHl,
Sacll, and Notl were
introduced in the gene encoding the BVDV-E2 protein.
Synthetic DNA is defined as a DNA that is made by synthesis, instead of being
isolated from a
natural source.
This does not necessarily mean that each and every nucleotide of the DNA is
synthesised. It is also
possible to modify an existing DNA by replacing part thereof by a part with
another nucleic acid
sequence using recombinant DNA technology. The result of this modification is
also considered to
be a synthetic DNA.
The synthetic DNA can be made in a number of different ways, all known in the
art. One useful
method for modifying a nucleotide sequence, is e.g. site-directed mutagenesis.
With this generally
known method, modifications are made deliberately at predetermined sites. It
is also possible to
replace small or longer fragments by fragments with an alternative sequence.
Many different
techniques for DNA-manipulation are currently available. One possible method
of replacing (parts
of) a naturally found sequence with a synthetically made sequence, is e.g. to
cut the nucleic acid
sequence with restriction enzymes to remove the sequence to be replaced,
followed by ligation of a
synthetically made fragment with the same restriction sites.
Alternatively, the whole gene can be replaced by synthetic DNA-fragments.
The synthetic BRSV E2 gene from which a splice site has been removed, and that
shows a minor
raise in GC-content as depicted in the overview of replacements in the
sequence of SEQ ID NO: 14
was shown to be very efficiently expressed. Therefore, in the most preferred
form of this
CA 02297507 2000-O1-31
_ g _
embodiment, the BVDV gene has the nucleotide sequence presented in the
overview of
replacements in the sequence of SEQ ID NO: 14.
It is clear, that the BVDV-gene, in order to be expressed, must be placed
under the control of a
promoter.
A large number of suitable promoters is known in the art, that are recognised
for their very efficient
level of expression. Such promoters are e.g. the Pseudorabies gX-promoter, the
Pseudorabies TK-
promoter, the Adenovirus Major Late promoter, the Retroviral Long Terminal
Repeat, the SV40
Early and Late promoters, the Mouse Cytomegalovirus immediate early (MCMVie1 )
promoter, the
Mouse Cytomegalovirus early (MCMVe1 ) promoter, the Human Cytomegalovirus
immediate early
(HCMVie1 ) promoter, and the BHV-gE promoter. All these promoters have been
described and are
known in the art for many years.
From these, the MCMVie1 promoter, the MCMVe1 promoter, the HCMVie1 promoter,
and the
BHV-gE promoter are preferred promoters.
Therefore, preferably the synthetic BVDV-gene according to the invention is
placed under the
control of one of the promoters of the group of promoters consisting of the
MCMVie1 promoter, the
MCMVe1 promoter, the HCMVie1 promoter, and the BHV-gE promoter.
Another embodiment of the invention relates to live attenuated carrier BHV
viruses carrying a
synthetic BVDV-gene according to the present invention.
As motivated above, BHV-viruses are the carriers of choice for BVDV-genes.
In a preferred form, as a recombinant BHV carrier, the BHV-1 virus is used.
This virus is a very
commonly found pathogen in cattle, also (as is the case with BVDV) causing
high economical
losses.
The most common manifestation of Bovine Herpes Virus-I infection is bovine
rhinotracheitis which
varies from a mild respiratory disease to a severe infection of the entire
respiratory tract.
From an economical point of view, Infectious Bovine Rhinotracheitis (IBR) is
also the most dramatic
manifestation of BHV-I infection. Morbidity rate in IBR is usually close to
100%.
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Therefore, using BHV-1 as the live attenuated recombinant carrier virus for
carrying and expressing
the BVDV-E2-gene is very efficient: vaccines based on such a live attenuated
recombinant virus
protect against both BVDV and BHV-1. Animals so vaccinated are protected
against the two most
frequently found causes of respiratory disease in cattle.
A large number of potential insertion sites can be used for the insertion of
the synthetic BVDV-gene
in the BHV carrier virus. The most suitable technique for such an insertion is
homologous
recombination, known in the art and frequently used. Preferably, the BVDV-gene
is cloned in a non-
essential BHV-gene, since in that case, a viable BHV-BVDV recombinant is
obtained that is not
disturbed in essential functions.
Several non-essential genes are known for BHV. The genes coding for the
(glyco)proteins gE, TK,
gl, gG, and US2 (=PrV 28k) are e.g. very suitable as integration sites.
Attenuated BHV-strains having deletions or insertions in such genes have been
described i.a. in
U.S.Patent No. 4,824,667, U.S.Patent No. 4,703,011, By Kit et al., in the
Veterinary Record 127:
363-364 (1990), in European patent Publication EP 0.326.127 and in European
Patent EP
0.587.805.
In a more preferred form, the BVDV-gene to be expressed is integrated in the
gE-gene of BHV.
In an equally more preferred form, the BVDV-gene to be expressed is integrated
in the gl-gene of
BHV-1.
A live attenuated recombinant BHV carrier virus according to the invention
may, next to one or
more BVDV-genes, comprise other genes encoding antigens from micro-organisms
or viruses that
are pathogenic for cattle.
Therefore, in a preferred form, the present invention provides live attenuated
recombinant BHV
carrier viruses comprising, next to a BVDV-gene, a gene encoding an antigen
from micro-
organisms or viruses that are pathogenic for cattle. Such genes are further
referred to as "additional
genes".
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In a more preferred form of this embodiment, the additional gene is chosen
from the group of cattle
pathogens, consisting of Bovine Rotavirus, Bovine Respiratory Syncytial Virus,
Parainfluenza type 3
virus, Bovine Paramyxovirus, Foot and Mouth Disease virus, and Pasteurella
haemolytica.
Also, an additional gene may be introduced into the live attenuated
recombinant BHV carrier virus
according to the invention, that encodes a cytokine. Several cytokines, e.g.
interferons are known to
play an important role as immune modulators. Thus it may be advantageous to
include genetic
information for this kind of molecules into said section.
Still another embodiment of the present invention refers to vaccines for the
protection of cattle
against virus infection, based upon live attenuated recombinant BHV carrier
virus expressing a
BVDV-gene, according to the invention.
Vaccines based thereon have the advantage that they mimic the natural
infection of not only BHV,
but to a large extend also of BVDV, as motivated above.
Therefore, they provide protection against more than one pathogen, in this
case against at least
BHV-infection and BVDV-infection.
Vaccination is in many cases at least a two-step process: a first immunisation
with an antigen
triggers the immune response, and a second immunisation, the booster, actually
enhances both the
speed and the strength of the immune response. After a first vaccination,
immunity against both the
carrier itself and the antigens encoded by the heterologous gene carried by
the carrier is triggered.
This is the result of the fact that the recombinant carrier infects a cell and
during viral replication
both the viral proteins and the heterologous protein of the carried gene are
expressed and become
presented at the cell membrane. There they are detected by the immune system.
A known problem
however, when using viruses as live recombinant carriers is the following:
antibodies against the
recombinant carrier raised during first immunisation prevent a successful
second round of infection,
a booster, with the recombinant carrier. Thus no expression of proteins after
boosting takes place,
and therefore the immune system will not see the encoded proteins a second
time. For the carrier
virus this problem can be circumvented by just giving a higher dose of virus
particles, since the viral
proteins are per se present on the virus particle. In this approach the virus
particles act as an
inactivated vaccine and as such stimulates the immune system. Infection is
however necessary for
the heterologous gene to be expressed. Therefore, since no second round of
infection occurs, the
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heterologous gene carried by the carrier is not expressed a second time. Thus
no booster
immunisation against the heterologous gene product will be generated.
It was now surprisingly found, that the BVDV E2 protein is incorporated into
the envelope of the
BHV-1 virus particles during the maturation of the virus. This is indeed
surprising since BVDV-E2
lacks any herpesspecific targeting signals and therefore could not be expected
to become
incorporated into the envelope of the BHV-1 particle. The unexpected
incorporation of BVDV-E2 in
the envelope of BHV means that the problem mentioned above can be
circumvented: a booster
immunisation with a high dose of BHV virus particles carrying the BVDV E2
protein on their
envelope causes a second immune response to be triggered against both the BHV
envelope
proteins and the BVDV protein on the envelope, without infection being
necessary.
Therefore, the vaccine according to the invention may also comprise a
recombinant BHV carrier
virus particle according to the invention as such, regardless if the particle
is still viable or not.
This embodiment of the invention thus relates also to inactivated Recombinant
BHV carrier virus
particles carrying the BVDV E2 glycoprotein on their surface.
Thus the invention relates to vaccines suitable for booster vaccination that
comprise inactivated
BHV carrier viruses according to the invention. Moreover, the invention
relates to the use of
inactivated BHV carrier viruses according to the invention for the manufacture
of vaccines for
booster vaccination against BVDV-infection.
The vaccine according to the present invention may comprise a pharmaceutically
acceptable
carrier. One possible carrier is a physiological salt-solution.
Other pharmaceutically acceptable carriers are for instance the solutions in
which adjuvants are
provided.
The invention also relates to methods for the preparation of vaccines
according to the invention.
Such methods comprise i.a. the admixing of the live attenuated BHV-recombinant
and/or the BHV-
recombinant virus particle and a pharmaceutically acceptable carrier.
If desired, an adjuvant and possibly one or more emulsifiers such as Tween~
and Span~ are also
incorporated in the live or inactivated vaccine according to the invention.
Suitable adjuvants are for
example vitamin-E acetate solubilisate, aluminum hydroxide, -phosphate or -
oxide, (mineral) oil
CA 02297507 2000-O1-31
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emulsions such as Bayol~ and Marcol52~ and saponins. Incorporation of the
antigens in Iscoms is
also a possible way of adjuvation.
It is advantageous to add a stabiliser to live or inactivated viruses,
particularly if a dry composition
of live viruses is prepared by lyophilisation. Suitable stabilisers are, for
example, SPGA (Bovamik et
al., J. Bacteriology 59 (1950) 509), carbohydrates (such as sorbitol,
mannitol, trehalose, starch,
sucrose, dextran or glucose), proteins (such as albumin or casein), or
degradation products thereof,
and buffers (such as alkali metal phosphates). If desired, one or more
compounds with adjuvant
activity as described above can also be added.
A vaccine according to the invention may be administered by intramuscular or
subcutaneous
injection or via intranasal, intratracheal, oral, cutane, percutane or
intracutane administration.
The natural route of infection is through the respiratory tract. Therefore, in
a preferred form, the
vaccine is formulated for intranasal administration. For the preparation of
such a vaccine, a
physiological salt solution is very attractive as a carrier, since it is
isotonic with mucosal fluid
Alternatively or in addition, the DNA isolated from the live attenuated BHV-
recombinant according to
the present invention can be directly administered. Vaccination methods using
naked DNA instead
of viruses are sufficiently known in the art, and have been described i.a. by
Cohen (Science 259:
1691-1692 (1993)), by Donnelly et al., The Immunologist 2: 20-26 (1993), by
Pardoll (Immunity 3:
165-169 (1995)) and by Montgomery (Current Biology 5: 505-510 (1994)).
The useful effective amount to be administered in a vaccine will vary
depending on the age, weight,
and mode of administration. A suitable vaccine dosage can for example comprise
about 103 - 10'°
pfu of the live recombinant per animal.
Another embodiment of the present invention relates to methods for the
preparation of vaccines
according to the invention. Such methods comprise admixing a live attenuated
BHV-recombinant or
an inactivated recombinant according to the present invention and a
pharmaceutically acceptable
carrier.
CA 02297507 2000-O1-31
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For the preparation of a live vaccine the BHV-I mutant according to the
present invention can be
grown on susceptible cells. Such susceptible cells are e.g. Madin Darby Bovine
Kidney cells
(MDBK-cells) or bovine embryonic cells.
The viruses thus grown can be harvested by collecting the tissue cell culture
fluids and/or cells. The
live vaccine may be prepared in the form of a suspension or may be
lyophilised.
Still another embodiment of the present invention refers to methods for the
preparation of live
attenuated BHV-recombinants according to the invention.
Such methods comprise bringing together in a suitable host cell BHV-DNA and a
vector comprising
the BVDV-gene to be expressed, placed under the control of a suitable promoter
and flanked by 3'
and 5' flanking regions that share homology with BHV-sequences, in order to
facilitate homologous
recombination.
This can be done by first transfecting the suitable host cell with the vector,
followed by infection with
the BHV-virus. Alternatively, both the vector and the isolated viral DNA may
transfected together
into a suitable host cell.
A generally acknowledged problem in the field of vaccination is the following:
the presence of
antibodies against a certain pathogen in the serum of a host mammal indicates
that the host has
been infected with the pathogen, either in a virulent form (e.g. through filed
infection) or an
attenuated form. When an animal becomes infected with BVDV this means that
antibodies
against all immunogenic BVDV-proteins are induced. It is however impossible to
discriminate
between field-infected mammals and mammals vaccinated with a vaccine strain,
regardless if
this vaccine strain is live or inactivated, since antibodies against the viral
proteins as such will be
abundantly present.
Surprisingly the recombinant BHV-virus carrying the BVDV-E2 gene according to
the present
invention offers a solution to this problem as follows:
The E2 gene is expressed in vivo in all BVDV field strains and induces anti-E2
antibodies in
infected animals. In addition, BVDV field strains induce significant amounts
of antibodies against
all other immunogenic BVDV-proteins. This implicates, that serum obtained from
mammals
infected with wild-type BVDV will always have antibodies against the E2-
protein as well as
.,
' ~ CA 02297507 2000-O1-31
- 14 -
antibodies against all other immunogenic BVDV-proteins. The BHV/BVDV-
recombinant according
to the present invention however can only induce antibodies against the BVDV
E2 protein.
Therefore mammals vaccinated with a BHV/BVDV-recombinant according to the
invention,
regardless the live or inactivated character, can only have antibodies against
the BVDV E2
protein in their serum. Thus sera obtained from animals vaccinated with the
BHV/BVDV-
recombinant according to the invention will only react with the BVDV E2
protein, whereas sera
obtained from animals infected with a BVDV field strain will contain
antibodies against all
immunogenic BVDV proteins.
Diagnostic tests based upon E2 protein and another immunogenic BVDV protein
can therefore
easily be used to discriminate between field-infection and vaccination with
the vaccine according
to the present invention.
The BHV/BVDV-recombinant according to the present invention thus is a very
suitable marker
vaccine, i.e. a vaccine strain that can be serologically discriminated from a
field strain.
A diagnostic test for the discrimination between vaccine strains and field
strains can be e.g. a
simple ELISA-test, or the incubation of a Western blot comprising the BVDV
proteins with serum
of mammals to be tested.
Thus, in still another embodiment, the invention relates to diagnostic tests
for the discrimination
between sera from mammals infected with BVDV field strains and from mammals
vaccinated with
a vaccine according to the invention.
The invention is further illustrated by way of the following Examples.
EXAMPLES
Example 1.
Construction of BVDV E2syn open reading frame (ORF) from synthetic
oligonucleotides.
The cDNA sequence of the E2-gene of the BVDV strain 68 was determined (see SEQ
ID NO: 14).
This sequence was used as a guiding sequence to compose a synthetic gene
called BVDV E2syn
ORF that has many convenient restriction enzyme cleavage sites which also will
facilitate the
construction of a whole range of smaller and larger deletion mutants. The
E2syn ORF does not
contain any splice donor consensus sequences but still codes for an E2 protein
with an amino acid
sequence identical to the BVDV strain 68 encoded E2 protein.
' ' ~ CA 02297507 2000-O1-31
- 15 -
To reach this goal a total of 13 fragments as given in SEQ ID NO: 1-13 were
obtained by mixing
equimolar amounts of complementary oligonucleotides in 10 mM tris-HCI, pH 7.5,
boiled for 5
minutes and then slowly cooled down to room temperature.
Fragments with SEQ ID NO: 1-7 were cloned adjacently in pSP73 yielding plasmid
pNH7. This was
done by cleaving pSP73 with Bglll and Xhol. Fragment 1 was ligated into this
DNA via its cohesive
ends resulting in Plasmid pNH1 which was cleaved with Stul and Xhol to receive
fragment 2. The
resulting plasmid pNH2 was cleaved with Pstl and Xhol and fragment 3 was
integrated to obtain
pNH3 which was cleaved with Hindlll and Xhol to construct pNH4 by integration
of fragment 4.
pNH4 was cleaved with Sful and Xhol and received fragment 5 resulting in pNH5
that was cleaved
with Bsu361 and Xhol for integration of fragment 6. The resulting plasmid pNH6
was cleaved with
Eagl and Xhol received fragment 7 to give plasmid pNH7.
Fragments A-F (SEQ ID NO: 8-13) were integrated in that order in pSP73
(Promega) to yield
pSPCF. This was done by cleaving pSP73 with Bglll and Xho. Fragment A was
ligated into this
DNA via its cohesive ends resulting in Plasmid pCA which was cleaved with
Bglll and BstXl to
receive fragment B. The resulting plasmid pCB was cleaved with Bgll and Sacll
and fragment C
was integrated to obtain pCC which was cleaved with Bglll and Apal to
construct pCD by
integration of fragment D. pCD was cleaved with Bglll and BsrGl and received
fragment E resulting
in pCE that was cleaved with Bglll and Accl for integration of fragment F to
obtain plasmid pCF.
Finally plasmid pNH7 was cleaved with BamHl and Xhol and received the purified
BamHl-Xhol
insert from plasmid pCF to yield plasmid pSPE2syn containing the reconstructed
BVDV E2 ORF.
Example 2
Construction and analysis of BHV-1 BVDV-E2-recombinan c
Cloning of the MCMV promoters ie1 and e1 in pAT-gD PLUS.
In order to test the expression of the E2 gene under various conditions, it
was decided to clone the
gene under the control of various alternative promoters.
CA 02297507 2000-O1-31
- 16 -
First of all, a multicloning site was inserted in pAT-gIV (Vector pAT-gIV has
been described
extensively in EP 0 663 403. It should be mentioned here that the vector pAT-
gIV is also referred to
as pAT-gD, due to the new nomenclature of the herpesvirus (glycoprotein-
)genes) between the
MCMVie2-polyA fragment and the gD-polyA fragment, and this new plasmid was
named pAT-gD
PLUS.
Starting with pAT-gD PLUS, two constructs were made:
Construction of Rp OMI~ Plasmid pAMB33 (borsch-Hasler et al., Proc. Natl.
Acid. Sci. 82 (1985)
8325-8329) was digested with Hpal and Xbal. Fragments were filled with Klenow-
polymerise and
isolated from agarose gels. A 1.4 kB fragment comprising the MCMV ie1 promoter
was obtained.
The plasmid pAT-gD PLUS was cleaved in its polylinker site with EcoRV. In this
site, the isolated
MCMV ie1 fragment was cloned, to yield pROMI (see Fig. 1 ).
Construction of~ROME~ Plasmid MM354, comprising the MCMV e1 promoter (sequence
of the
Murine Cytomegalovirus early 1 gene is present in the EMBL Gene Bank at
Heidelberg, Germany)
was digested with Smal and Hindlll. Fragments were filled with Klenow-
polymerise and isolated
from agarose gels. A 1.1 kB fragment comprising the MCMV e1 promoter was
obtained. The
plasmid pAT-gD PLUS was cleaved in its polylinker site with EcoRV. In this
site, the isolated MCMV
e1 fragment was cloned, to yield pROME (See Fig. 2).
Cloning of Rn OME.~
To provide a signal peptide for correct transport and processing of BVDV E2,
the pestivirus signal
sequence of CSFV strain Alfort (See SEQ ID NO: 16) was ligated into pROME (see
above) after
cleavage with BamHl and Kpnl to give plasmid pROMEsig.
Cloning of the BVDV E2~m ORF into r~ROMEsia Plasmid pROMEsig was cleaved with
Kpnl and
Notl and used for the integration of the BVDV E2syn ORF cleaved out of plasmid
pSPE2syn with
Kpnl and Notl. The resulting plasmid was named pROME2syn.
Integration of cROME2gyn into BHV-1 MDBK cells were transfected with 5 mg
pROME2syn and
2.5 mg of purified DNA of gD negative virus BHV-1/80-221 (Fehler et al.,
J.Virol. 66 (1992) 831-
CA 02297507 2000-O1-31
- 17 -
839). Transfected cells were shocked with glycerol 4 hours after addition of
the DNA. After
development of CPE the cell culture supernatant was titrated on MDBK cells.
Under these
conditions only genotypically gD positive virions can grow productively and
form plaques on
monolayers. At 3 days p.i. virions from single plaques were isolated and DNA
from MDBK cells was
purified, cleaved with Hindlll, size separated by agarose gel electrophoresis
and transferred to
nitrocellulose filters which were hybridised to 32P-labeled probes either from
the gD ORF, the IacZ
ORF, the E2syn ORF or sequences downstream the E2syn ORF presented for
homologous
recombination. The results demonstrated that the isolated virions lack the f3-
galactosidase
sequences and that the BVDV E2syn ORF was integrated. The resulting virus BHV-
1/E2syn was
plaque purified twice and used for further charactererization.
Identification and characterization of BHV 1 ex~se~ BVDV F2
To demonstrate expression by recombinant BHV-1 and to identify the BVDV E2syn
ORF encoded
gene product proteins from wild type BHV-1 strain Schonboken (Fig. 3, lanes 1
and 3) and
recombinant BHV-1/E2syn (Fig. 3, lanes 2 and 4), infected MDBK cells were
analysed by
immunoblotting using an immune serum from a BVDV strain 68 infected calf
(panel a) or a rabbit
serum directed against BHV-1 glycoprotein D. The anti-BVDV serum did not
specifically react with
proteins from BHV-1 strain Schonboken infected cells but bound to a 53 kDa
apparent molecular
mass protein among BHV-1/E2syn infected cell proteins. This apparent molecular
mass
corresponds very well to the 53 kDa apparent molecular mass reported for the
E2 glycoprotein
expressed in BVDV infected cells. Binding of the anti-gD serum to the 72 and
68 kDa apparent
molecular mass forms of BHV-1 gD (panel b) demonstrates comparable infection
with BHV-1 strain
Schonboken and BHV-1 /E2syn, respectively.
Fig. 4 demonstrates incorporation of the BHV-1/E2syn expressed E2 glycoprotein
into recombinant
virions. For this purpose MDBK cells were infected with BHV-1/E2syn in the
presence of 35S-
methionine. Proteins from lysed cells (lane 1 ) and purified virions (lane 2)
were immunoprecipitated
using the anti BVDV serum and bound proteins were visualised by fluorography
after size
separation in SDS-polyacrylamide gels. The result shows that the E2
glycoprotein is associated
with BHV-1/E2syn virions.
CA 02297507 2000-O1-31
- 18 -
LEGENDS TO THE FIGURES
Fig. 1 shows a map of pROMI
Fig. 2 shows a map of pROME
Fig. 3 demonstrates expression by recombinant BHV-1 and identifies the BVDV
E2syn ORF
encoded gene product.
MDBK cells were infected with wild type BHV-1 strain Schonboken (Fig. 3, lanes
1 and 3) and
recombinant BHV-1/E2syn (Fig. 3, lanes 2 and 4), and their protein content
analysed by
immunoblotting using an immune serum from a BVDV strain 68 infected calf
(panel a) or a rabbit
serum directed against BHV-1 glycoprotein D (panel b).
Fig. 4 demonstrates incorporation of the BHV-1/E2syn expressed E2 glycoprotein
into recombinant
virions. MDBK cells were infected with BHV-1/E2syn in the presence of 35S-
methionine. Proteins
from lysed cells (lane 1 ) and purified virions (lane 2) were
immunoprecipitated using the anti BVDV
serum and bound proteins were visualised by fluorography after size separation
in SDS-
polyacrylamide gels. The result shows that the E2 glycoprotein is associated
with BHV-1/E2syn
virions.
CA 02297507 2000-04-26
19
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: A1CZ0 NOVEL N.V.
(ii) TITLE OF INVE1QTION: SYNTHETIC GENE OF BOVINE VIRAL DIARRHOEA VIRUS
(iii) NUMBER OF SEQiJENCES: 16
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: FETHERSTONHAUGH & CO.
(B) STREET: I?Ø BOX 2999, STATION D
(C) CITY: OT'.CAWA
(D) STATE: ONT
(E) COUNTRY: CANADA
(F) ZIP: K1P 5Y6
(v) COMPUTER READABLE FORM:
(A) MEDIUM T7.'PE: Floppy disk
(B) COMPUTER: IBM PC: compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: ASCII (text)
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: CA 2,297,507
(B) FILING DATE: 31-JAN-2000
(C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: EP 99200304.6
(B) FILING DATE: 02-FEB-1999
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: E'ETHERSTONHAUGH & CO.
(B) REGISTRATION NUMBER:
(C) REFERENCE;/DOCKET NUMBER: 23804-561
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE;: (613)-235-4373
CA 02297507 2000-04-26
(B) TELEFAX: 0'013)-232-8440
(2) INFORMATIO1V FOR SEQ ID NO.: 1:
(i) SEQUENCE C13ARACTERISTICS
(A) LENGTH: 92
(ii) MOLECULAR TYPE: DNA
(A) ORGANISM: bovine viral diarrhoea virus
(ix) FEATURE:
10 (A) NAME/KEY: mist recomb
(B) LOCATION: (1)..(4)
(D) OTHER INFORMATION: "5' sticky end: the
indicated sequence is aingle
stranded; no nuc:Leotides present on the
complementary strand"
(ix) FEATURE:
(A) NAME/KEY: mist recomb
(B) LOCATION: (5)..(57)
(D) OTHER INFORMATION: "double stranded part of the
20 oligonucleotide"
(ix) FEATURE:
(A) NAME/KEY: mist recomb
(B) LOCATION: (58)..(61)
(D) OTHER INFORMATION: "3' sticky end: the
indicated sequence is .single
stranded; nucleotides TOGA are not present in this
fragment and only indicate that the complementary
strand contains :nucleotides AGCT"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO.: 1:
GATCTGGTAC CACTTAGACT GCAAACCTGA ATACTACTAT GCCATAGCCA AGAATGATAG 60
AGTCGGCCTA CTAGGAGCTG ,zIAGGCCTCTC GA 92
CA 02297507 2000-04-26
21
(2) INFORMATION FOR SEQ ID NO.: 2:
(i) SEQUENCE C;HARACTERISTICS
(A) LENGTH: 101
(ii) MOLECULAR TYPE: DNA
(A) ORGANISM: bovine viral diarrhoea virus
(ix) FEATURE:
(A) NAME/KEY: misc recomb
(B) LOCATION: (1)..(97)
(D) OTHER INFORMATION: "double stranded part of the
nucleotide"
(ix) FEATURE:
(A) NAME/KEY: misc recomb
(B) LOCATION: (98)..(101)
(D) OTHER INFORMATION: "3' sticky end: the
indicated sequence is single
stranded; nuclectides TCGA are not present in this
fragment and only indicate that the complementary
strand contains nucleotides AGCT"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO.: 2:
CCTTACCACT GTTTGGAAGG AATACTCACC TGAAATGACG CTGGAAGACA CAATGGTCAT 60
AGCCTCCTGC AGAGAAGGCA AATTTACATA CCGCTCCTCG A 101
(2) INFORMATION FOR SEQ ID NO.: 3:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 114
(ii) MOLECULAR TYPE: DNA
(A) ORGANISM: bovine viral diarrhoea virus
(ix) FEATURE:
3 0 (A) NAME/KEY: misc recomb
(B) LOCATION: (1)..(4)
CA 02297507 2000-04-26
22
(D) OTHER INFORMATION: "5' sticky end: the
indicated sequence is single
stranded; the nucleotides TCGA are not present in
this fragment anal only indicate that the
complementary strand contains nucleotides AGCT"
(ix) FEATURE:
(A) NAME/KEY: mist recomb
(B) LOCATION: (5)..(110)
(D) OTHER INFORMATION: "double stranded part of the
oligonucleotide" ..
(ix) FEATURE:
(A) NAME/KEY: mist recomb
(B) LOCATION: (111)..(114)
(D) OTHER INFORMATION: "3' sticky end: the
indicated sequence is single
stranded: nucleotides TCGA are not present in this
fragment and only indicate that the vcomplementary
strand contains nucleotides AGCT"
2O (xi) SEQUENCE DESCRIPTION: SEQ ID NO.: 3:
TGCAGAGAAG GCAAATTTAC ATACCGCTCA AGATGCACAA GGGAAGCTAG ATATCTTGCA 60
ATTTTGCATT CAAGAGCCTT ACCGACCAGC GTGGTATTTG AGAAGCTTAC TOGA 114
(2) INFORMATION FOR SEQ ID NO.: 4:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 62
(ii) MOLECULAR TYPE: DNA
(A) ORGANISM: bovine viral diarrhoea virus
(ix) FEATURE:
30 (A) NAME/KEY: mist recomb
(B) LOCATION: (1)..(4)
CA 02297507 2000-04-26
23
(D) OTHER INFORMATION: "5' sticky end: the
indicated sequence is single
stranded; no nucleotides present on the
complementary strand"
(ix) FEATURE:
(A) NAME/KEY: misc recomb
(B) LOCATION: (5)..(58)
(D) OTHER INFORMATION: "double stranded part of the
oligonucleotide"
(ix) FEATURE:
(A) NAME/KEY: misc recomb
(B) LOCATION: (59)..(62)
(D) OTHER INFORMATION: "3' sticky end: the
indicated sequence is single
stranded; nuclec>tides TCGA are not present in this
fragment and only indicate that the complementary
strand contains nucleotides AGCT"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO.: 4:
2 O AGCTTTTCGA GGGCCAAAAG CAAGAGGACA CGGTTGAGAT GGATGACAAC TTCGAATCTC 60
GA 62
(2) INFORMATIC)N FOR SEQ ID NO.: 5:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 108
(ii) MOLECULAR TYPE: DNA
(A) ORGANISM: bovine viral~diarrhoea virus
(ix) FEATURE:
(A) NAME/KEY: misc recomb
3 O (B) LOCATION: (1)..(2)
CA 02297507 2000-04-26
24
(D) OTHER INFORMATION: "5' STICKY END: THE
INDICATED SEQUENCE IS .SINGLE
STRANDED; NO NUCLEOTIDES PRESENT ON THE
COMPLEMENTARY STRAND"
(ix) FEATURE:
(A) NAME/KEY: misc recomb
(B) LOCATION: (3)..(104)
(D) OTHER INFORMATION: "DOUBLE STRANDED PART OF THE
OLIGONUCLEOTIDE"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO.: 5:
CGAATTTGGA CTCTGCCCAT GCGACGCCAA GCCCGTAGTA AAGGGGACTT TCAATACAAC 60
ACTGCTAAAT GGACCGGCTT 'TCCAGATGGT ATGCCCCTTA GGGCTCGA 108
(2) INFORMATION FOR SEQ ID NO.: 6:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 96
(ii) MOLECULAR TYPE: DNA
(A) ORGANISM: bovine viral diarrhoea virus
2 O (ix) FEATURE:
(A) NAME/KEY: misc difference
(B) LOCATION: (1)..(3)
(D) OTHER INFORMATION: "5' sticky end: the
indicated sequence is single
stranded; no nucleotides present on the complementary
strand"
(ix) FEATURE:
(A) NAME/KEY: misc recomb
(B) LOCATION: (4)..(92)
30 (D) OTHER INFORMATION: "double stranded part of the
oligonucleotide"
CA 02297507 2000-04-26
(ix) FEATURE:
(A) NAME/KEY: mist recomb
(B) LOCATION: (93)..(96)
(D) OTHER INFORMATION: "3' sticky end: the
indicated sequence is single
stranded; nucleotides TCGA are not present in this
fragment and only indicate that the complementary
strain contain nucleotides AGCT"
ZO (xi) SEQUENCE DESCFCIPTION: SEQ ID NO.: 6:
TTAGGGTGGA CAGGGACCGT GAGCTGCATG TTAGCTAATA GGGATACCCT AGATACAGCA 60
GTAGTGCGGA CGTATAGGAG ATATCGGCCG TCTCGA 96
(2) INFORMATION FOR SEQ ID NO.: 7:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 131
(ii) MOLECULAR TYPE: DNA
(A) ORGANISM: bovine viral diarrhoea virus
(ix) FEATURE:
2 0 (A) NAME/KEY: mist recomb
(B) LOCATION: (1)..(4)
(D) OTHER INFORMATION: r'5' sticky end: the
indicated sequence is single
stranded; no nucleotides present on the
complementary strand"
(ix) FEATURE:
(A) NAME/KEY: mist recomb
(B) LOCATION: (5)..(127)
(D) OTHER INFORMATION: "double stranded part of the
oligonucleotide"
(ix) FEATURE:
CA 02297507 2000-04-26
26
(A) NAME/KEY: misc recomb
(B) LOCATION: (128)..(131)
(D) OTHER INFORMATION: "3' sticky end: the
indicated sequence is single
stranded nucleotides TCGA are not present in this
fragment and only indicate that the complementary
strand contains nucleotides AGCT"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO.: 7:
GGCCGTTCCC TTACAGGCAA GACTGCATCA CCCAGAAGGT TCTGGGAGAG GATCTCTATA 60
ACTGCATTCT TGGAGGAAAC TGGACCTGCA TAACTGGAGA CCAACTACAA TACTCAGGAG 120
GATCCACTCG A 131
(2) INFORMATION FOR SEQ ID NO.: 8:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 122
(ii) MOLECULAR TYPE: DNA
(A) ORGANISM: bovine viral diarrhoea virus
(ix) FEATURE:
2 0 (A) NAME/KEY: misc recomb
(B) LOCATION: (1)..(4)
(D) OTHER INFORMATION: "5' sticky end: the
indicated sequence is single
stranded: no nucleotides present in the
complementary strand"
(ix) FEATURE:
(A) NAME/KEY: misc recomb
(B) LOCATION: (5)..(120)
(D) OTHER INFORMATION: "double stranded part of the
30 oligonucleotide"
(ix) FEATURE:
CA 02297507 2000-04-26
27
(A) NAME/KEY: misc recomb
(B) LOCATION: (121)..(122)
(D) OTHER INFORMATION: "3' sticky end: the
indicated sequence is single
stranded; nucleotides AG are not present in this
fragment and only indicate that the complementary
strand contains nucleotides TC"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO.: 8:
IO GATCTACGGA TCCATTGAAT CCTGCAAATG GTGCGGCTTT AAATTCCAAA GAAGCGAGGG 60
GTTACCACAC TACCCCATTG GCAAGTGCAG GCTGAAGAAT GAGACTGGCT ACAGATTAGT 120
AG 122
(2) INFORMATION FOR SEQ ID NO.: 9:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 91
(ii) MOLECULAR TYPE: DNA
(A) ORGANISM: bovine viral diarrhoea virus
(ix) FEATURE:
2 0 (A) NAME/KEY: misc recomb
(B) LOCATION: (1)..(4)
(D) OTHER INFORMATION: "5' sticky end: the
indicated sequence is single
stranded; no nucleotides present on the
complementary strand"
(ix) FEATURE:
(A) NAME/KEY: misc recomb
(B) LOCATION: (5)..(87)
(D) OTHER INFORMATION: "double stranded part of the
30 oligonucleotide"
(ix) FEATURE:
CA 02297507 2000-04-26
28
(A) NAME/KEY: misc recomb
(B) LOCATION: (88)..(91)
(D) OTHER INFORMATION: "3' sticky end: the
indicated sequence is single
stranded nucleotides GTAC are not present in this
fragment and on)_y indicate that the complementary
strand contains nucleotides CATG"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO.: 9:
lO GATCTGGTAG ACGACACCTC TTGCAATATA GGAGGCGTGG CGATAGTACC ACAGGGGATG 60
GTAAAATGCA AGATAGGAGA CACAATTGTA C 91
(2) INFORMATION FOR SEQ ID NO.: 10:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 84
(ii) MOLECULAR TYPE: DNA
(A) ORGANISM: bovine viral diarrhoea virus
(ix) FEATURE:
(A) NAME/KEY: misc recomb
20 (B) LOCATION: (1)..(4) ..
(D) OTHER INFORMATION: "5' sticky end: the
indicated sequence is single
stranded: no nucleotides present on the
complementary strand"
(ix) FEATURE:
(A) NAME/KEY: misc recomb
(B) LOCATION: (5)..(80)
(D) OTHER INFORMATION: "double stranded part of the
oligonucleotide"
3 O (ix) FEATURE:
(A) NAME/KEY: misc recomb
CA 02297507 2000-04-26
29
(B) LOCATION: (81)..(84)
(D) OTHER INFORMATION: "3' sticky end: the
indicated sequence is single
stranded; no nuc:Leotides present on the
complementary strand"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO.: 10:
GATCTCTGTA CAGGTCATAG CTCTTGACAC CAAACTTGGG CCTATGCCCT GCAAGCCATA 60
TGAGATCATT TCAAGCGAGG c;GCC 84
1~
(2) INFORMATION FOR SEQ ID NO.: 11:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 76
(ii) MOLECULAR TYPE: DNA
(A) ORGANISM: bovine viral diarrhoea virus
(ix) FEATURE:
(A) NAME/KEY: misc_recomb
(B) LOCATION: (1)..(4)
(D) OTHER INFORMATION: "5' sticky end: the
20 indicated sequence is single
stranded; no nucleotides present on the
complementary strand"
(ix) FEATURE:
(A) NAME/KEY: misc recomb
(B) LOCATION: (5)..(74)
(D) OTHER INFORMATION: "double stranded part of the
oligonucleotide"
(ix) FEATURE:
(A) NAME/KEY: misc recomb
30 (B) LOCATION: (75)..(76)
CA 02297507 2000-04-26
(D) OTHER INFORMATION: "3' sticky end; the
indicated sequence is single
stranded; no nucleotides present on the
complementary strand"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO.: 11:
GATCTCGGGC CCGTAGAAAA GACGGCATGC ACCTTCAACT ACACGAGGAC ATTAAAGAAC 60
AAGTACTTTG AGCCGC 76
10 (2) INFORMATION FOR SEQ ID NO.: 12:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 114
(ii) MOLECULAR TYPE: DNA
(A) ORGANISM: bovine viral diarrhoea virus
(ix) FEATURE:
(A) NAME/KEY: misc recomb
(B) LOCATION: (1)..(4)
(D) OTHER INFORMATION: "5' sticky end: the
indicated sequence is single
20 stranded; no nucleotides present on the
complementary strand"
(ix) FEATURE:
(A) NAME/KEY: misc feature
(B) LOCATION: (5)..(110)
(D) OTHER INFORMATION: "double stranded part of the
oligonucleotide"
(ix) FEATURE:
(A) NAME/KEY: misc recomb
(B) LOCATION: (111)..(114)
30 (D) OTHER INFORMATION: "3' sticky end: the
indicated sequence is single
CA 02297507 2000-04-26
31
stranded; no nucleotides present on the
complementary strand"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO.: 12:
GATCTACCGC GGGACAGCTA C:TTCCAGC:AA TATATGCTAA AAGGAGAATA TCAATACTGG 60
TTTGACCTGG AGGTAACCGA C;CATCACCGG GATTACTTGG CCGAATCCAT ATTA 114
(2) INFORMATION FOR SEQ ID NO.: 13:
(i) SEQUENCE CHARACTERISTICS
lO (A) LENGTH: 115
(ii) MOLECULAR TYPE: DNA
(A) ORGANISM: bovine viral diarrhoea virus
(ix) FEATURE:
(A) NAME/KEY: misc recomb
(B) LOCATION: (1)..(4)
(D) OTHER INFORMATION: "5' sticky end: the
indicated sequence is single
stranded; no nucleotides present on the
complementary strand"
2 O (ix) FEATURE:
(A) NAME/KEY: misc recomb
(B) LOCATION: (5)..(111)
(D) OTHER INFORMATION: "double stranded part of the
oligonucleotide"
(ix) FEATURE:
(A) NAME/KEY: misc recomb
(B) LOCATION: (112)..(115)
(D) OTHER INFORMATION: "3' sticky end: the
indicated sequence is single
30 stranded; nucleotides TCGA are not present in this fragment and
only indicate that the compelemtary strand contains nucleotide AGCT"
CA 02297507 2000-04-26 -
32
{xi) SEQUENCE DESCRIPTION: SEQ ID NO.: 13:
GATCTCCATA TTAGTGGTCG TCGTCGCTCT CCTGGGCGGC AGATACGTCC TCTGGCTACT 60
GGTCACATAC ATGGTTCTAT CAGAACAAAA GGTCTTAGGG TGAGCGGCCG CTCGA 115
(2) INFORMATION FOR SEQ ID NO.: 14:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 1122
(ii) MOLECULAR TYPE: DNA
(A) ORGANISM: bovine viral diarrhoea virus
(ix) FEATURE:
(A) NAME/KEY: misc feature
(B) LOCATION: (1)..(1122)
(D) OTHER INFORMATION: "cDNA sequence of the BVDV
strain 68"
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: (1)..(1122)
(D) OTHER INFORMATION:
2O (ix) FEATURE:
(A) NAME/KEY: misc difference
(B) LOCATION: (107)..(108)
(D) OTHER. INFORMATION: replace by "AG"
(ix) FEATURE:
(A) NAME/KEY: misc difference
(B) LOCATION: (140)..{141)
(D) OTHER INFORMATION: replace by "CC"
(ix) FEATURE:
(A) NAME/KEY: misc difference
30 (B) LOCATION: (152)..(153)
CA 02297507 2000-04-26
33
(D) OTHER INFORMATION: replace by "GC"
(ix) FEATURE:
(A) NAME/KEY: misc difference
(B) LOCATION: (155)..(156)
(D) OTHER INFORMATION: replace by "AA"
(ix) FEATURE:
(A) NAME/KEY: misc difference
(B) LOCATION: (173)..(174)
(D) OTHER INFORMATION: replace by "GA"
(ix) FEATURE:
(A) NAME/KEY: misc difference
(B) LOCATION: (230)..(231)
(D) OTHER. INFORMATION: replace by "GC"
(ix) FEATURE:
(A) NAME/KEY: misc difference
(B) LOCATION: (242)..(243)
(D) OTHER INFORMATION: replace by "AG"
(ix) FEATURE:
(A) NAME/KEY: misc difference
(B) LOCATION: (245)..(246)
(D) OTHER INFORMATION: replace by "AG"
(ix) FEATURE:
(A) NAME/KEY: misc difference
(B) LOCATION: (251)..(252)
(D) OTHER INFORMATION: replace by "TC"
(ix) FEATURE:
(A) NAME/KEY: misc difference
(B) LOCATION: (257)..(258)
(D) OTHER INFORMATION: replace by "GC"
(ix) FEATURE:
CA 02297507 2000-04-26
34
(A) NAME/KEY: misc difference
(B) LOCATION: (278)..(279)
(D) OTHER INFORMATION: replace by "TT"
(ix) FEATURE: w
(A) NAME/KEY: misc difference
(B) LOCATION: (419)..(420)
(D) OTHER INFORMATION: replace by "GC"
(ix) FEATURE:
(A) NAME/KEY: misc difference
(B) LOCATION: (473)..(474)
(D) OTHER INFORMATION: replace by "GA"
(ix) FEATURE:
(A) NAME/KEY: misc difference
(B) LOCATION: (477)..(478)
(D) OTHER INFORMATION: replace by "TC"
(ix) FEATURE:
(A) NAME/KEY: misc difference
(B) LOCATION: (479)..(480)
2 0 (D) OTHER INFORMATION: replace by "GC"
(ix) FEATURE:
(A) NAME/KEY: misc difference
(B) LOCATION: (482)..(483)
(D) OTHER INFORMATION: replace by "CG"
(ix) FEATURE:
(A) NAME/KEY: misc difference
(B) LOCATION: (512)..(513)
(D) OTHER INFORMATION: replace by "AG"
(ix) FEATURE:
30 (A) NAME/KEY: misc difference
(B) LOCATION: (515)..(516)
CA 02297507 2000-04-26
(D) OTHER INFORMATION: replace by "AG"
(ix) FEATURE:
(A) NAME/KEY: misc difference
(B) LOCATION: (524)..(525)
(D) OTHER INFORMATION: replace by "GA"
(ix) FEATURE:
(A) NAME/KEY: misc difference
(B) LOCATION: (542)..(543)
10 (D) OTHER INFORMATION: replace by "GC"
(ix) FEATURE:
(A) NAME/KEY: misc difference
(B) LOCATION: (566)..(567)
(D) OTHER INFORMATION: replace by "GC"
(ix) FEATURE:
(A) NAME/KEY: misc difference
(B) LOCATION: (575)..(576)
(D) OTHER INFORMATION: replace by "GA"
(ix) FEATURE:
20 (A) NAME/KEY: misc difference
(B) LOCATION: (599)..(600)
(D) OTHER INFORMATION: replace by "GA"
(ix) FEATURE:
(A) NAME/KEY: misc difference
(B) LOCATION: (602)..(603)
(D) OTHER INFORMATION: replace by "CC"
(ix) FEATURE:
(A) NAME/KEY: misc difference
(B) LOCATION: (614)..(615)
30 (D) OTHER. INFORMATION: replace by "GC"
(ix) FEATURE:
CA 02297507 2000-04-26
36
(A) NAME/KEY: misc difference
(B) LOCATION: (617)..(618)
(D) OTHER INFORMATION: replace by "AA"
(ix) FEATURE:
(A) NAME/KEY: misc difference
(B) LOCAT:LON: (623)..(624)
(D) OTHER INFORMATION: replace by "GC"
(ix) FEATURE:
(A) NAME/KEY: misc difference
(B) LOCATION: (626)..(627)
(D) OTHER INFORMATION: replace by "GC"
(ix) FEATURE:
(A) NAME/KEY: misc difference
(B) LOCATION: (635)..(636)
(D) OTHER INFORMATION: replace by "TT"
(ix) FEATURE:
(A) NAME/KEY: misc difference
(B) LOCATION: (644)..(645)
(D) OTHER INFORMATION: replace by "GC"
(ix) FEATURE:
(A) NAME/KEY: misc difference
(B) LOCATION: (677)..(678)_.
(D) OTHER INFORMATION: replace by "GC"
(ix) FEATURE:
(A) NAME/KEY: misc difference
(B) LOCATION: (637)..(638)
(D) OTHER INFORMATION: replace by "GC"
(ix) FEATURE:
(A) NAME/KEY: misc difference
(B) LOCATION: (767)..(768)
CA 02297507 2000-04-26
37
(D) OTHER INFORMATION: replace by "AA"
(ix) FEATURE:
(A) NAME/KEY: misc difference
(B) LOCATION: (770)..(771)
(D) OTHER INFORMATION: replace by "GC"
(ix) FEATURE:
(A) NAME/KEY: misc difference
(B) LOCAT:CON: (884)..(885)
(D) OTHER INFORMATION: replace by "GC"
(ix) FEATURE:
(A) NAME/KEY: misc difference
(B) LOCATION: (914)..(915)
(D) OTHER INFORMATION: replace by "AC"
(ix) FEATURE:
(A) NAME/KEY: misc difference
(B) LOCATION: (917)..(918)
(D) OTHER INFORMATION: replace by "AG"
(ix) FEATURE:
(A) NAME/KEY: misc difference
(B) LOCATION: (929)..(930)
(D) OTHER INFORMATION: replace by "CG"
(ix) FEATURE:
(A) NAME/KEY: misc difference
(B) LOCATION: (931)..(932)
(D) OTHER. INFORMATION: replace by "CG"
(ix) FEATURE:
(A) NAME/KEY: misc difference
(B) LOCATION: (933)..(934)
(D) OTHEP, INFORMATION: replace by "GG"
(ix) FEATURE:
CA 02297507 2000-04-26
38
(A) NAME/KEY: mist difference
(B) LOCATION: (938)..(939)
(D) OTHER INFORMATION: replace by "GC"
(ix) FEATURE:
(A) NAME/KEY: mist difference
(B) LOCATION: (998)..(999)
(D) OTHER INFORMATION: replace by "CC"
(ix) FEATURE:
(A) NAME/KEY: mist difference
(B) LOCATION: (1025)..(1026)
(D) OTHER INFORMATION: replace by "AA"
(ix) FEATURE:
(A) NAME/KEY: mist difference
(B) LOCATION: (1039)..(1040)
(D) OTHER INFORMATION: replace by "GC"
(ix) FEATURE:
(A) NAME/KEY: mist difference
(B) LOCATION: (1042)..(1043)
2 0 (D) OTHER INFORMATION: replace by "GC"
(ix) FEATURE:
(A) NAME/KEY: mist difference
(B) LOCATION: (1046)..(1047)
(D) OTHER. INFORMATION: replace by "TC"
(ix) FEATURE:
(A) NAME/KEY: mist difference
(B) LOCATION: (1058)..(1059)
(D) OTHER INFORMATION: replace by "GC"
(ix) FEATURE:
3 0 (A) NAME/KEY: mist difference
(B) LOCATION: (1061).(1062)
CA 02297507 2000-04-26
39
(D) OTHER INFORMATION: replace by "GC"
(ix) FEATURE:
(A) NAME/1CEY:mist fference
di
(B) LOCAT:LON:(1070)..(1071)
(D) OTHER INFORMATION: replaceby "TC"
(ix) FEATURE:
(A) NAME/KEY: mist fference
di
(B) LOCATION: (1077)..(1078)
(D) OTHER INFORMATION: replaceby "GC"
(ix) FEATURE:
(A) NAME/KEY: mist fference
di
(B) LOCATION: (1112)..(1113)
(D) OTHER INFORMATION: replace by "AG"
(xi) SEQUENCEDESCRIPTION : NO.:
SEQ 14:
ID
CAC TTA GACTGCAAA CC'TGAA TAC TAT ATAGCCAAG AATGAT 48
TAC GCC
His Leu AspCysLys ProGlu Tyr Tyr IleAlaLys AsnAsp
Tyr Ala
1 5 10 15
2 AGA GTC GGCCTACTA GG.AGCT GAA CTT ACTGTTTGG AAGGAA 96
O GGC ACC
Arg Val GlyLeuLeu GlyAla Glu Leu ThrValTrp LysGlu
Gly Thr
20 25 30
TAC TCA CCTGAAATG ACGCTG GAA ACA GTCATAGCC TCGTGC 144
GAC ATG
Tyr Ser ProGluMet ThrLeu Glu Thr ValIleAla SerCys
Asp Met
35 40 45
AGA GAA GGTAAGTTT ACATAC CGC AGG ACAAGGGAA GCTAGA 192
TCA TGC
Arg Glu GlyLysPhe ThrTyr A.rg Arg ThrArgGlu AlaArg
Ser Cys
30 50 55 60
TAT CTT GCAATTTTG CATTCA AGA TTA ACCAGTGTG GTATTT 240
GCC CCG
Tyr Leu AlaIleLeu HisSer Arg Leu ThrSerVal ValPhe
Ala Pro
65 70 75 80
GAA AAA CTTTTTGAG GGGCAA P,AG GAG ACGGTCGAG ATGGAT 288
CAA GAC
Glu Lys LeuPheGlu GlyGln L~ys Glu ThrValGlu MetAsp
Gln Asp
85 90 95
40 GAC AAC TTCGAATTT GGACTC fGC TGC GCCAAGCCC GTAGTA 336
CCA GAC
Asp Asn PheGluPhe GlyLeu Cys Cys AlaLysPro ValVal
Pro Asp
100 105 110
AAG GGG ACTTTCAAT AC'.AACA CTG AAT CCGGCTTTC CAGATG 384
CTA GGA
Lys Gly ThrPheAsn ThrThr Leu Asn ProAlaPhe GlnMet
Leu Gly
115 1.20 125
CA 02297507 2000-04-26
GTA TGC CCC TTA GGG TGG ACA GGG ACC GTG AGC TGT ATG TTA GCT AAT 432
Val Cys Pro Leu Gly Trp Thr Gly Thr Val Ser Cys Met Leu Ala Asn
130 135 140
AGG GAT ACC CTA GAT ACA GCA GTA GTG CGG ACG TAT AGG AGG TAT AGA 480
Arg Asp Thr Leu Asp Thr Ala Val Val Arg Thr Tyr Arg Arg Tyr Arg
145 150 155 160
lO CCA TTC CCT TAC AGG CAA GAC TGC ATC ACC CAA AAA GTT CTG GGG GAG 528
Pro Phe Pro Tyr Arg Gln Asp Cys Ile Thr Gln Lys Val Leu Gly Glu
165 170 175
GAT CTC TAT AAC TGT ATT CTT GGA GGA AAC TGG ACC TGT ATA ACT GGG 576
Asp Leu Tyr Asn Cys Ile Leu Gly Gly Asn Trp Thr Cys Ile Thr Gly
180 185 190
GAC CAA CTA CAA TAC TCA GGA C;GC TCT ATT GAA TCC TGT AAG TGG TGT 624
Asp Gln Leu Gln Tyr Ser Gly Gly Ser Ile Glu Ser Cys Lys Trp Cys
20 195 2:00 205
GGT TTT AAA TTC CAA AGA AGT GAG GGG TTA CCA CAC TAC CCC ATT GGC 672
Gly Phe Lys Phe Gln Arg Ser Glu Gly Leu Pro His Tpr Pro Ile Gly
210 215 220
AAG TGT AGG CTG AAG AP.T GAG ACT GGC TAC AGA TTA GTA GAC GAC ACC 720
Lys Cys Arg Leu Lys Ann Glu Thr Gly Tyr Arg Leu Val Asp Asp Thr
225 23.0 235 240
3O TCT TGC AAT ATA GGA GGT GTG GCG ATA GTA CCA CAG GGG ATG GTA AAG 768
Ser Cys Asn Ile Gly Gl.y Val Ala Ile Val Pro Gln Gly Met Val Lys
245 250 255
TGT AAG ATA GGA GAC AC;A ATT C~TA CAG GTC ATA GCT CTT GAC ACC AAA 816
Cys Lys Ile Gly Asp Thr Ile Val Gln Val Ile Ala Leu Asp Thr Lys
260 265 270
CTT GGG CCT ATG CCC TGC AAG C;CA TAT GAG ATC ATT TCA AGT GAG GGG 864
Leu Gly Pro Met Pro Cys Lys E?ro Tyr Glu Ile Ile Ser Ser Glu Gly
40 275 2 80 285
CCC GTA GAA AAG ACG GC;A TGT ACC TTC AAC TAC ACG AGG ACA TTA AAG 912
Pro Val Glu Lys Thr A~.a Cys Thr Phe Asn Tyr Thr Arg Thr Leu Lys
290 295 300
AAT AAA TAC TTT GAG CC;C AGA GAC AGT TAC TTC CAG CAA TAT ATG CTA 960
Asn Lys Tyr Phe Glu Pro Arg Asp Ser Tyr Phe Gln G1n Tyr Met Leu
305 37.0 315 320
AAA GGA GAG TAT CAA TAC TGG TTT GAC CTG GAG GTA ACT GAC CAT CAC 1008
Lys Gly Glu Tyr Gln Tyr Trp Phe Asp Leu Glu Val Thr Asp His His
325 330 335
CGG GAT TAC TTG GCC GAG TCC ATA TTA GTG GTG GTG GTA GCT CTC CTG 1056
Arg Asp Tyr Leu Ala Glu Ser :Cle Leu Val Val Val Val Ala Leu Leu
340 345 350
GGT GGT AGA TAC GTG C'.CC TGG TTA CTG GTC ACA TAC ATG GTT CTA TCA 1104
Gly Gly Arg Tyr Val Leu Trp Leu Leu Val Thr Tyr Met Val Leu Ser
355 :360 365
CA 02297507 2000-04-26
41
GAA CAA AAA GTC TTA GG~~ 1122
Glu Gln Lys Val Leu Gly
370
(2) INFORMATION FOR SEQ ID NO.: 15:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 374
(ii) MOLECULAR TYPE: POLYPEPTIDE
(A) ORGANISM: bovine viral diarrhoea virus
(xi) SEQUENCE DESCRIPTION: SEQ ID NO.: 15:
His Leu Asp Cys Lys Pro Glu Tyr Tyr Tyr Ala Ile Ala Lys Asn Asp
1 5 10 15
Arg Val Gly Leu Leu Gly Ala Glu Gly Leu Thr Thr Val Trp Lys Glu
20 25 30
Tyr SerProGluMet ThrLeu GluAspThrMet ValIleAla SerCys
35 40 45
Arg GluGlyLysPhe Th.rTyr ArgSerArgCys ThrArgGlu AlaArg
50 55 60
Tyr LeuAlaIleLeu HisSer ArgAlaLeuPro ThrSerVal ValPhe
65 70 75 80
Glu LysLeuPheGlu GlyGln LysGlnGluAsp ThrValGlu MetAsp
85 90 95
3 Asp AsnPheGluPhe Gl.yLeu C:ysProCysAsp AlaLysPro ValVal
0
100 105 110
Lys GlyThrPheAsn ThrThr LeuLeuAsnGly ProAlaPhe GlnMet
115 120 125
Val CysProLeuGly TrpThr GlyThrValSer CysMetLeu AlaAsn
130 135 140
Arg AspThrLeuAsp ThrAla ValValArgThr TyrArgArg TyrArg
40 145 1'.i0 155 160
Pro PheProTyrArg G7_nAsp (:ysIleThrGln LysValLeu GlyGlu
165 170 175
Asp LeuTyrAsnCys I:.eLeu GlyGlyAsnTrp ThrCysIle ThrGly
180 185 ~ 190
Asp GlnLeuGlnTyr SeerGly GlySerIleGlu SerCysLys TrpCys
195 ;?00 205
50
Gly PheLysPheGln ArgSer GluGlyLeuPro HisTyrPro IleGly
210 215 220
Lys CysArgLeuLys AanGlu 'ThrGlyTyrArg LeuValAsp AspThr
225 2:30 235 240
CA 02297507 2000-04-26
42
Ser Cys Asn Ile Gly Gly Val Ala Ile Val Pro Gln Gly Met Val Lys
245 250 255
Cys Lys Ile Gly Asp Thr Ile Val Gln Val Ile Ala Leu Asp Thr Lys
260 265 270
Leu Gly Pro Met Pro Cys Lys Pro Tyr Glu Ile Ile Ser Ser Glu Gly
275 280 285
Pro Val Glu Lys Thr Ala Cys Thr Phe Asn Tyr Thr Arg Thr Leu Lys
290 295 300
Asn Lys Tyr Phe Glu Pro Arg Asp Ser Tyr Phe Gln Gln Tyr Met Leu
305 310 315 320
Lys Gly Glu Tyr Gln Tyr Trp Phe Asp Leu Glu Val Thr Asp His His
325 330 335
Arg Asp Tyr Leu Ala Glu Ser I:Le Leu Val Val Val Val Ala Leu Leu
340 345 350
Gly Gly Arg Tyr Val Leu Trp Leu Leu Val Thr Tyr Met Val Leu Ser
355 360 365
Glu Gln Lys Val Leu G1!T
370
(2) INFORMATIO1J FOR SEQ ID NO.: 16:
(i) SEQUENCE CIiARACTE1.~ISTICS
(A) LENGTH: 70
(ii) MOLECULAR TYPE: DNA
(A) ORGANISM: bovine viral diarrhoea virus
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: (10)..(69)
(ix) FEATURE:
(A) NAME/KEY: misc-feature
(B) LOCATION: (1)..(70)
4 0 (D) OTHER INFORMATION: "Pestivirus signal sequence
of CSFV strain Alfort
(xi) SEQUENCE DESCRIPTION: SEQ ID NO.: 16:
GGATCCACC ATG GCC CTG TTG GCT TGG GCG GTG ATA ACA ATC TTG CTG TAC 51
Met Ala Leu Leu Ala Trp Ala Val Ile Thr..Ile Leu Leu Tyr
1 5 10
CA 02297507 2000-04-26
43
CAG CCT GTA GCA GGG TAC; C 7 0
Gln Pro Val Ala Gly Tyr
15 20
