Note: Descriptions are shown in the official language in which they were submitted.
CA 02418780 2003-02-28
FIELD OF THE INVENTION
The present invention pertains to the field of recombinant mammalian vectors,
particularly to
novel PRRSV-related sequences in vectors for the immunization of animals
against porcine
reproductive and respiratory syndrome virus (PRRSV).
BACKGROUND
Strains of PRRSV
Porcine reproductive and respiratory syndrome virus (PRRSV) has been found to
be the
causative agent of porcine reproductive and respiratory syndrome (PRRS), an
economically
important viral disease that affects swine worldwide. Although the clinical
syndromes associated
with PRRSV infection are similar in North America and Europe, strains from the
two continents
are distinct and DNA sequence analysis of both North American and European
strains has
revealed high genomic variations (Mardassi et al., (1994) J. Gen. Virol.
75:681-685; Mardassi et
al., (1994) J. Clin. Microbiol. 32:2197-2203; Meng et al., (1995) Arch. Virol.
140:745-755;
Murtaugh et al. , (1995) Arch. Virol. 140:1451-1460). The prototype European
strain of PRRSV
is the Lelystad virus (LV). This virus was described in EP 0 587 780 B1.
North American and European strains of PRRSV display a high degree of
variability in their
ORF 2, 3, S, and 7 coding regions with less than 60% amino acid identities
(Mardassi et al.,
(1995) Arch. Virol. 140:1405-1418; Meng et al., (1995) J. Gen. Virol. 76:3181-
3188; Meng et
al., (1995) Arch. Virol. 140:745-755; Murtaughetal., (1995) Arch. Virol.
140:1451-1460).
Unique PRRSV strains have been isolated in Quebec (Dea et al., (1992) Can.
Vet. J. 33:801-808;
Mardassi et al., (1994) Can. J. Vet. Res. 58:55-64; Mardassi et al., (1994) J.
Gen. Virol. 75:681-
685). One such strain was adapted to grow in cell culture and is known as the
Quebec reference
cytopathic strain IAF-Klop (Mardassi et al., (1995) Arch. Virol. 140:1405-
1418).
PRRSV
2S PRRSV was first isolated in the Netherlands (Wensvoort et al., (1991) Vet.
Q. 13:121-130) in the
early 1990s, being associated with a similar syndrome in most of the pig
producing countries
within the next three year-period (reviewed in Dea et al. , (2000) Arch.
Virol. 145:659-688).
PRRSV belongs to the recently recognized Arteriviridae family in the genus
Arterivirus, order
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CA 02418780 2003-02-28
Nidovirales along with equine arteritis virus (EAV), simian hemorrhagic fever
virus (SHFV) and
lactate dehydrogenase elevating virus (LDV) (Cavanagh, (I997) Arch. Virol.
142:629-633; de
Vries et al., (1997) Seminars in Virology 8:33-47). The disease is clinically
characterized by
reproductive disorders in sows and gilts and respiratory problems affecting
pigs of all ages
(Goyal, (1993) J. Vet. Diagnostic Investigations 5:656-664).
The viral genome consists of a positive single-stranded RNA molecule of
approximately 1S kb in
length, composed of nine open reading frames (ORFla, ORFIb, ORF2a, ORF2b and
ORF3-7).
The ORF1, which represents nearly 75% of the viral genome, encodes for
proteins with apparent
replicase and polymerase. The ORFs 5 to 7 encode for the envelope glycoprotein
GPS (25-26
kDa), the non-glycosylated membrane protein M (18-19 kDa) and the nucleocapsid
protein N
(14-15 kDa), which are the three major structural proteins of PRRSV. These
PRRSV structural
proteins are closely associated both in the infected cells and in the viral
particles, the GP5 and M
proteins being associated in the form of heterodimers. The structural nature
of the ORF3 product,
a highly glycosylated protein with an apparent M~ of 42 kDa, is still being
debated, in view of the
apparently conflicting data on its presence on the virions of North American
and European
PRRSV strains. The ORFs 2a and 4 encode for the GPZ and GP4 glycoproteins,
with respective
Mr of 29 and 31 kDa, and have been identified as minor structural
glycoproteins of the virion.
Recently, it has been demonstrated that the ORF2 is a bicistronic gene, an
additional 10-kDa
protein being encoded by a second ORF, named ORF2b, which start codon is only
6 nucleotides
downstream of the adenine of the ORF2a start codon (Dea et al., (2000) Arch.
Virol. 145:659-
688; Snijder & Meulenberg, (1998) J. Gen. Virol. 79:961-979; Wu et al., (2001)
Virology
297:183-191 ).
Vaccines
Anti-PRRSV swine immunization practices have used live attenuated viral
vaccines to mimic a
natural infection, however a serious disadvantage of such vaccines is their
pathogenicity in
immunosuppressed recipients exposed to environmental stress, such as the poor
housing and
over-crowding that are often prevalent in intensive animal raising operations.
This can be of
great concern in veterinary medicine where clinical outbreaks are sometimes
reported shortly
after prophylactic immunization. These vaccines also require special handling
to maintain
viability and to avoid tissue culture contaminants.
Administration of live modified vaccines is also problematic because such
vaccines may result in
virus persistence, which in turn contributes to the generation of mutants
through selective
immune pressure on the resident variants. Persistently infected animals may
eventually shed
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CA 02418780 2003-02-28
newly generated mutants, particularly in the case of unstable pathogens such
as RNA viruses,
and these mutants may be responsible for new outbreaks.
Production and purification of large quantities of viral particles for use in
whole viral inactivated
vaccines or their immunogenic structural proteins is economically unfeasible
for low yield
viruses such as PRRSV. Antigenic viral peptides may be expressed recombinantly
in bacteria,
yeast, or even mammalian cells and harvested for use in immunizations, however
they often
require extensive treatment to ensure appropriate antigenicity.
Circulating antibodies in PRRSV-infected pigs responsible for viral
neutralization in cell cultures
are mainly directed against GPS (Gonin etal., (1999) J. Vet. Diagnostic
Investigation 11:20-26).
Immunization experiments of mice with E.rcherichia coli-expressed GST-ORFS
recombinant
fusion protein, as well as with purified PRRSV, induced specific anti-GPS
neutralizing
monoclonal antibodies (Pirzadeh & Dea, (1997) J. Ger.. Virol. 73:1867-1873;
Zhang et al.,
(1998) Veterinary Microbiology 63:125-136). Furthermore, genetic immunization
of pigs, with
plasmidic DNA expressing the ORFS gene under the control of the human
cytomegalovirus
immediate-early promotor/enhancer, not only triggered the production of
neutralizing antibodies
to PRRSV but also conferred protection against development of clinical disease
and lung lesions
following an intratracheal challenge with a high dose of PRRSV (Pirzadeh &
Dea, (1998) J. Gen.
Virol. 79:989-999; Gagnon et al., (2001) Adv Exp Med Biol. 494:225-31).
However, DNA immunization is apparently not sufficient to inhibit virus
persistence and
shedding in the respiratory tract of PRRSV challenged pigs (Pirzadeh & Dea,
(1998) J. Gen.
Virol. 79:989-999). Although these constructs were able to induce a specific
immune response,
circulating antibody titres were transient and low. On the other hand, when
the E. coli-expressed
GST-ORFS recombinant fusion protein was used as an immunogen prior to a
challenge with the
pathogenic virus, the disease was more severe despite the development of high
titres (> 1:2048)
of non-neutralizing antibodies to GPS. This observation suggests that an
antibody-dependent
enhancement (ADE) phenomenon could be involved where such anti-GPS antibodies
may
facilitate PRRSV infection through the attachment of the immune complexes to
Fc receptors
present at the surface of alveolar macrophages (Molitor et al., (1997) Vet.
Microbiol. 55:265-
276; Pirzadeh & Dea, (1998) J. Gen. Virvl. 79:989-999). These findings also
suggest that the
amounts of GPS synthesized in the infected cells, as well as the conformation
of the mature
protein which rnay be influenced by the type of oligosaccharide side chains
present on the
molecule, are apparently crucial to trigger an effective humoral immune
response to PRRSV.
Due to the correlation apparent amongst protection, clinical course of the
disease, and
seroneutralizing antibody titres (Loemba et al., (1996) Arch. Virol. 141:751-
761; Pirzadeh &
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Dea, (1998) J. Gen. Virol. 79:989-999; Vezina et al.. (1996) Can. J. Vet. Res.
60:94-99), an
increase in the efficacy of genetic immunization against antigenic
determinants of the major GPS
envelope-associated glycoprotein could provide improved response.
Considering the increased incidence of PRRSV infection through the world, a
need remains for
an effective vaccine against PRRSV infection. The vaccine should be safe for
use in swine,
including pregnant sows and suckling, unweaned, and growing pigs. As well, a
test is needed for
the serological diagnosis of PRRSV infection that can differentiate between
vaccination and
infection with naturally occurring strains of PRRSV.
This background information is provided for the purpose of making known
information believed
by the applicant to be of possible relevance to the present invention. No
admission is necessarily
intended, nor should be construed, that any of the preceding information
constitutes prior art
against the present invention. Publications referred to throughout the
specification are hereby
incorporated by reference in their entireties in this application.
SUMMARY OF THE INVENTION
The present invention is directed to synthetic genes encoding proteins of
porcine reproductive
and respiratory syndrome virus and uses thereof. In accordance with an aspect
of the present
invention, there is provided an isolated nucleic acid molecule comprising a
nucleotide sequence
that encodes the amino acid sequence of a porcine reproductive and respiratory
syndrome virus
(PRRSV) polypeptide, said nucleotide sequence having at least one non-
preferred codon replaced
by a preferred codon.
In accordance with another aspect of the present invention, there is provided
a vector comprising
a nucleic acid molecule of the invention.
In accordance with another aspect of the present invention, there is provided
a host cell
genetically engineered with a nucleic acid molecule of the invention or with a
vector of the
invention.
In accordance with another aspect of the present invention, there is provided
a process for
producing recombinant a host cell capable of expressing a PRRSV polypeptide or
fragment
thereof, comprising genetically engineering said cell with a nucleic acid
molecule of the
invention, or with a vector of the invention.
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In accordance with another aspect of the present invention, there is provided
a process for
producing a PRRSV polypeptide or a fragment thereof, comprising culturing a
host cell of the
invention, and recovering the polypeptide encoded by the nucleotide sequence
from the culture.
In accordance with another aspect of the present invention, there is provided
an isolated
polypeptide having an amino acid sequence encoded by a nucleic acid molecule
of the invention,
or produced by culturing a host cell of the invention, and recovering the
polypeptide encoded by
the nucleotide sequence from the culture.
In accordance with another aspect of the present invention, there is provided
a use of a nucleic
acid molecule of the invention or a vector of the invention, in the
preparation of a composition.
In accordance with another aspect of the present invention, there is provided
a composition,
comprising a nucleic acid molecule of the invention or a vector of the
invention and a carrier or
adjuvant.
In accordance with another aspect of the present invention, there is provided
a kit, comprising a
composition of the invention, and a storage container suitable for containing
said composition.
I5 In accordance with another aspect of the present invention, there is
provided a use of a
composition of the invention for immunization of a pig in need thereof.
In accordance with another aspect of the present invention, there is provided
a method of making
antibodies against one or more PRRSV proteins comprising administering to an
animal a nucleic
acid molecule of the invention, or a vector of the invention or a composition
of the invention,
collecting at least one blood sample at a suitable times post-administration,
and isolating a serum
fraction containing antibodies against one or more PRRSV proteins.
In accordance with another aspect of the invention, there is provided
antibodies against one or
more PRRSV proteins prepared by a method comprising administering to an animal
a nucleic
acid molecule of the invention, or a vector of the invention or a composition
of the invention,
collecting at least one blood sample at a suitable times post-administration,
and isolating a serum
fraction containing antibodies against one or more PRRSV proteins.
In accordance with another aspect of the present invention, there is provided
an isolated serum
composition suitable for immunization of a pig against PRRSV, comprising an
effective amount
of semi-purified blood serum obtained from a mammal inoculated with a
composition of the
invention.
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In accordance with another aspect of the present invention, there is provided
a use of a serum
obtained from a mammal inoculated with a composition of the invention to
immunize a pig in
need thereof.
In accordance with another aspect of the present invention, there is provided
a kit for detecting in
a sample an antibody that specifically recognizes a PRRSV polypeptide or an
antigenic fragment
thereof, comprising a nucleic acid molecule of the invention, or a vector of
the invention, or a
host cell of the invention, which is capable of expressing one or more PRRSV
polypeptides
and/or one or more antigenic fragments thereof.
In accordance with another aspect of the invention, there is provided a
diagnostic composition
comprising a nucleic acid molecule of the invention, or a vector of the
invention and optionally a
diluent or carrier.
In accordance with another aspect of the invention, there is provided a primer
capable of
specifically hybridizing a nucleic acid molecule of the invention.
In accordance with another aspect of the invention, there is provided a method
for detecting in a
sample obtained from a pig, the presence of a nucleic acid molecule of the
invention, or a portion
thereof, comprising: obtaining a sample from a pig; contacting said sample
with at least one
primer of the invention under hybridizing conditions; and determining the
presence of a
hybridizing nucleic acid sequence in said sample, wherein said pig was
previously immunized
with a composition comprising a nucleic acid molecule of the invention, and
said nucleotide
sequence is not complimentary to a nucleic acid molecule of the invention.
In accordance with another aspect of the invention, there is provided a kit
for detecting in a
sample obtained from a pig, the presence of a polynucleotide specific to the
nucleic acid
molecule of the invention or a fragment thereof, comprising at least one
primer of the invention,
and a suitable container for containing said at least one primer.
Various other objects and advantages of the present invention will become
apparent from the
detailed description of the invention.
BRIEF DESCRIPTION OF THE FIGURES
Figure 1. Figure 1 shows sequence of a codon-optimized ORFS polynucleotide
that encodes
fragments of a modified ORFS polypeptide from the IAF-94-287 strain of PRRSV.
CA 02418780 2003-02-28
Figure 2. Figure 2 shows the sequence of the 603-by ORFS of the IAF-Klop
strain of PRRSV
(GenBank Accession No U64928) and that of the synthesized (synORFS) with the
deduced
amino acid sequence. Changed codons for synORFS are underlined. Deduced amino
acid
sequences from both wtORFS and synORFS genes are identical.
Figure 3. Figure 3 shows the immunofluorescence staining of 293 cells at 48 h
post-transfection
with the pRc/CMV2 (control), pRc/CMV2/wtORFS (WT) or pRc/CMV2/synORFS (SYNT)
recombinant plasmids. Expression of GPS of PRRSV (strain IAF-Klop) was
confirmed by
indirect immunofluorescence (IIF) following incubation in the presence of the
rabbit anti-a5
monospecific serum.
Figure 4. Figure 4 shows co-expression in the simian MARC-I45 cells of the
reporter gene
GFPq (green fluorescent protein) inserted upstream of the wtORFS or synORFS
genes in the
constructed hAdVs. (Left) Intensity of fluorescence spontaneously released by
the GFPq protein
in MARL-145 cell monolayers co-infected with AdVCMV/tTA and
hAdV/TR5/DC/GFPq/wtORFS at a multiplicity of infection (moi) of 100 after 24,
48 and 70h of
incubation, respectively. (Right) Intensity of fluorescence spontaneously
released by the GFPq
protein in MARC-145 cell monolayers co-infected with AdVCMV/tTA and
hAdV/TR5/DC/GFPq/synORFS at a moi of 100 after 24 , 48 and 70 h of incubation,
respectively.
Figure S. Figure 5 shows the expression in the simian MARC-145 cells of the
recombinant GPS
envelope glycoprotein of PRRSV (strain IAF-Klop) by hAdVs expressing the
wtORFS or
synORFS genes. MARL-145 cell monolayers were co-infected with AdCMV/tTA and
hAdV/TR5/DC/GFPq/wtORFS (lane WT) or hAdV/TR5/DC/GFPq/synORFS (lane SYNT) at a
moi of 100 PFU, then fixed with cold acetone and washed twice with PBS after
24, 48 and 70 h
of incubation, respectively, to eliminate spontaneous GFPq fluorescence.
Expression of GPS of
PRRSV was confirmed by specific IIF following incubation in the presence of
the rabbit anti-a5
monospecific serum.
Figure 6. Both panels of Figure 6 show the radioimmunoprecipitation of GPS
protein from
lysates of 293rtTA cells infected 48 h earlier with either
hAdVlTRS/DC/GFPq/wtORFS (lane
WT) or hAdV/TR5/DC/GFPq/synORFS (lane SYNT). The immune complexes obtained
after
incubation in the presence of rabbit anti-a5 monospecific serum were adsorbed
on protein A-
sepharose beads, then analysed by SDS-PAGE and revealed by fluorography and
autoradiography. The major structural proteins of the PRRSV (proteins N, M and
GPS, having
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CA 02418780 2003-02-28
relative molecular weights of 14, 19 and 24-26 kDa respectively), could be
immunoprecipitated
from lysates of PRRSV-infected cells (lane V, left panel only) but not from
lysates of mock-
infected cells (-). The ratio of lysate cpm loaded in each lane is shown at
the bottom on each
panel. '°C-radiolabelled molecular weight size standards (in kDa) were
migrated in lane L.
Figure 7. Figure 7 shows the increased anti-GPS humoral immune response of
pigs immunized
with synORFS or control vaccine mixtures as revealed by Western blot. Piglets
were injected
intradermally twice at 32 day-interval with either AdCMVS/tTA alone (tTA), a
1:5 mixture of
hAdV/TR5/DC/GFPq/wtORFS + AdCMV/tTA (wtORFS) or a 1:5 mixture of
hAdV/TR5/DC/GFPq/synORFS + AdCMV/tTA (synORFS), as described in the materials
and
methods section. They were challenged intranasally four weeks after the
booster injection with
105 TCIDso of the homologous IAF-Klop strain of PRRSV. Western blot strips
were prepared
using sucrose-gradient purified PRRSV (IAF-Klop strain) as antigen. The
reactivity profiles of
serum from all 9 pigs are illustrated for serum collected 10 days (panel A)
and 21 days post-
challenge (panel B). The positive control (+) corresponds to the reactivity of
the a5 rabbit
monospecific hyperimmune serum with the sucrose gradient purified virus
(Mardassi et al.,
1996). These data are also presented in Table 4: (-) no GPS band; (w) weak
band; (+) moderate
or strong band.
Figure 8. Figure 8 shows the sequence of (top line) a completely codon-
optimized ORFS
sequence (synORFS) of the IAF-Klop strain of PRRSV. This nucleotide sequence
is compared to
(middle line) a partially codon-optimized ORES polynucleotide (synORFS
variant) based on a
modified (non-wild-type) ORFS sequence, and (bottom line) the 603-by wild-type
ORFS
sequence (GenBank Accession No U64928). Compared to the synORF5 sequence, only
the
replaced nucleotides are shown for the variant and wild-type sequences.
Variant nucleotides that
are underlined differ from those used in the synORFS sequence, and variant
nucleotides that are
in boxes alter the amino acid expressed relative to the synORFS (and wild-
type) sequence. The
synORFS variant is the partially codon-optimized version and the deduced amino
acid sequence
differs from the wild type in having C48Y, A63S, W155L and G183A. For example
the amino
acid at position 183 is G in the protein expressed from the wild-type ORFS and
synORFS
sequences, and is A in the protein expressed from the modified ORFS and
synORF5 variant
sequences. Deduced amino acid sequences from the wtORFS gene and synORFS
polynucleotide
are identical.
Figure 9. Figure 9 shows the expression in 293 cells of the partially codon-
optimized ORFS
sequence of Figure 8. The synORFS variant sequence and an unoptimized wild-
type sequence
(ORFS sequence of the IAF-Klop strain) were each cloned into a pVAX plasmid
vector
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CA 02418780 2003-02-28
(Invitrogen Canada Inc., Burlington, Ontario) and transfected into 293 cells
as described
previously. Cells were fixed after 48 h of incubation and expression of the
transfected ORFS
polynucleotides was confirmed by specific IIF following incubation in the
presence of the rabbit
anti-a5 monospecific serum. Expression of the ORFS proteins was increased for
both vectors
compared to a control vector (pVAX), and greatest for the synORFS variant.
Figure 10. Figure 10 shows the sequence of the 537-by ORF4 (GenBank accession
number
AF003345) of the IAF-Klop strain of PRRSV, and that of a codon-optimized ORF4
polynucleotide. The nucleotides substituted in order to optimize the codons of
synORF4 are
shown. Deduced amino acid sequences from both wtORF4 and synORF4
polynucleotides are
identical.
Figure 11. Figure 11 shows the sequence of the 525-by ORF6 of the IAF-Klop
strain of PRRSV
(GenBank Accession No U64928) and that of a codon-optimized ORF6
polynucleotides.
Deduced amino acid sequences from both wtORF6 and synORFb polynucleotides are
identical.
Figure 12. Figure shows a hydrophilicity plot for the GP5 protein (encoded by
the wtORFS
polynucleotide) of 4 different strains of PRRSV.
Tables
Table 1. North American PRRSV strains and their GenBank accession numbers.
Table 2. Examples of preferred codons for optimal expression in mammalian
cells.
Table 3a. Table 3a shows the frequency of codon occurrence in humans (H) and
in the ORFS
protein (GPs) of the IAF-Klop strain of PRRSV. The frequencies of the
individual codons are
shown as percentages for each of the degenerately encoded amino acids, as well
as the number of
each amino acid for GPS (in parentheses). The most prevalent codon is shown
underlined in
bold.
Table 3b. Table 3b shows the frequency of codon occurrence in humans (H) and
in the ORF4
gene (encoding the GPI protein) of the IAF-Klop strain of PRRSV. The
frequencies of the
individual codons are shown as percentages for each of the degenerately
encoded amino acids, as
well as the number of each amino acid for GPI (in parentheses). The most
prevalent codon is
shown underlined in bold.
Table 3c. Table 3c shows the frequency c>f codon occurrence in humans (H) and
in the ORF6
gene (encoding the M protein) of the IAF-Klop strain of PRRSV. The frequencies
of the
CA 02418780 2003-02-28
individual codons are shown as percentages for each of the degenerately
encoded amino acids, as
well as the number of each amino acid for the M protein (in parentheses). The
most prevalent
codon is shown underlined in bold.
Table 4. Post-challenge antibody response to immunization with vaccines
expressing either the
wild type or synthetic codon-optimized PRRSV ORFS gene.
Table 5. Oligonucleotide primers used in PCR amplification of the synthetic
ORFS (synORFS of
IAF-Klop, Figure 2, SEQ ID NO: 1)
DETAILED DESCRIPTION OF THE INVENTION
The present invention resides in the discovery that use of a codon-optimized
nucleic acid
sequence encoding a PRRSV protein to immunize swine results in increased
expression of the
protein product and an enhanced immune response in swine.
The present invention provides for codon-optimized PRRSV ORF nucleic acid
sequences and
expression constructs and their use to immunize swine against PRRSV. Fragments
of a full
length ORF gene that encode fragments of the full-length PRRSV protein can
also be constructed
and used.
Definitions
Unless defined otherwise, all technical and scientific terms used herein have
the same meaning as
commonly understood by one of ordinary skill in the art to which this
invention belongs.
Reference can be made to, for example, " Immunobiology" (5th ed. CA Janeway et
al. Garland
Publishing, c2001) regarding immunological terms and concepts in the art. The
following terms
and abbreviations are used throughout the specification and in the claims.
As used herein, "nucleic acid molecule" refers to a polymeric form of
nucleotides of any length,
both to ribonucleic acid sequences and to deoxyribonucleic acid sequences. In
principle, this
term refers to the primary structure of the molecule; thus, this term includes
double and single
stranded DNA, as well as double and single stranded RNA, and modifications
thereof. This term
may be used interchangeably with the terms "polynucleotide" and "nucleic acid
sequence".
As used herein, "expression construct" encompasses, for example, expression
cassettes and
expression vectors, and refers to a genetic construct containing one or more
nucleic acid
sequences coding for all or part of one or more gene products, which are
capable of being
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CA 02418780 2003-02-28
transcribed. The transcript may be translated into a protein, but it need not
be. Thus, in certain
embodiments, expression includes both transcription of the nucleic acid
sequence and translation
of the mRNA into a gene product. In other embodiments, expression only
includes transcription
of the nucleic acid sequence. The nucleic acid sequences coding for all or
part of one or more
gene products can be a subunit of an expression construct.
"Operably linked" refers to a juxtaposition wherein the components so
described are in a
relationship permitting them to function in their intended manner. A control
sequence "operably
linked" to a coding sequence is ligated in such a way that expression of the
coding sequence is
achieved under conditions compatible with the control sequences.
"Control sequence" refers to polynucleotide sequences which are necessary to
effect the
expression of coding and non-coding sequences to which they are ligated. The
nature of such
control sequences differs depending upon the host organism; in prokaryotes,
such control
sequences generally include promoter, ribosomal binding site, and
transcription termination
sequence; in eukaryotes, generally, such control sequences include promoters
and transcription
termination sequence. The term "control sequences" is intended to include, at
a minimum,
components whose presence can influence expression, and can also include
additional
components whose presence is advantageous, for example, leader sequences and
fusion partner
sequences.
As used herein, "porcine reproductive and respiratory syndrome", or "PRRS"
refers to the
disease syndromes Mystery Swine Disease, Mystery Pig Disease, Mystery Disease,
Mystery
Reproductive Syndrome, swine plague, New Pig Disease, Wabash syndrome, abortus
blau, Blue
Eared Pig Disease, Porcine Epidemic Abortion and Respiratory Syndrome (PEARS),
swine
infertility and respiratory syndrome (SIRS), Epidemic Late Abortion of Swine
(ELAS),
Hyperthermia, Anorexia and Abortion Syndrome of the Sow (HAASS), Porcine
Arterivirus, the
disease caused by the Iowa strain of PRRSV, and closely-related variants of
these diseases which
have appeared and which will appear in the future. The symptoms of this
syndrome are well
known in the art, and include but are not limited to reproductive disorders
and respiratory
problems (Goyal et al., (1993) J Vet Diagn Invest 5:656-664). Severe
reproductive disorder is
characterized by mummified fetuses, stillbirths, late term abortions,
premature farrowings and an
increase in neonatal mortality associated with weak-born piglets. Severe
respiratory distress is
more pronounced in newborn and nursing pigs but can affect pigs of all ages
and is characterized
by chronic pneumonia cause by interstitial pneumonia, in severe case mouth
breathing can be
observed. Also, the PRRS disease has the appearance of an influenza like
syndrome. Other
symptoms may occurs like conjunctivitis, blue discoloration of the skin,
fever, lethargy, poor
12
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appetite, sneezing, vomiting and in rare case central nervous system signs may
develop (Goyal et
al., (1993) J Vet Diagn Invest 5:656-664).
As used herein, "porcine reproductive and respiratory virus (PRRSV)" refers to
the causative
agent of a porcine reproductive and respiratory syndrome, as described above.
As used herein, a "vaccine" refers to an agent used to vaccinate (immunize)
swine, i.e. to protect
swine, against PRRS resulting from infection by PRRS virus. "Vaccine" can
additionally mean
an agent whereby, after administration of the agent to an unaffected swine,
and subsequent
exposure to a PRRSV, lesions in the lung or symptoms of the syndrome do not
appear or are not
as severe as in infected, untreated swine. An unaffected swine is one that has
either not been
exposed to a PRRSV, or that has been exposed to a PRRSV but is not showing
symptoms of the
syndrome. An affected swine is one that is showing symptoms of the syndrome.
As used herein, "ORF" refers to an open reading frame of a nucleotide sequence
encoding a
PRRSV protein. That is, in the context of the present invention an ORF is a
sequence of a
nucleic acid molecule that encodes a PRRSV polypeptide. An ORF polynucleotide
sequence can
be a wild-type sequence or a modified sequence. It is further understood that
the term ORF can
refer to the full length of an ORF gene or ORF coding sequence, or less than
the full length of
these sequences, i.e. to fragments or subsequences of an ORF gene. PRRSV
proteins are known
in the art by various names, including as ORF proteins distinguished by
including a numeral after
the prefix "ORF". e.g. ORFS, ORF6, ORF2a, etc.
As used herein, "codon" refers to a sequence of three consecutive nucleotides,
of either
ribonucleic acid or deoxyribonucleic acid, which constitutes the instruction
for incorporation of a
specific amino acid in a specific position in a polypeptide chain during
protein synthesis. An
amino acid may be encoded by more than one codon and frequency of codon-usage
varies
between species. A codon that is rarely used by a species is herein referred
to as a "non-
preferred codon", and a codon that is most frequently used by a species is
herein referred to as a
"preferred" codon.
As used herein, "codon optimization" refers to the design of a nucleic acid
sequence encoding a
known amino acid sequence so that one or more non-preferred codons is replaced
by a preferred
codon whereby the designed (optimized) nucleic acid sequence encodes the same
known amino
acid sequence as the unoptimized sequence.
13
CA 02418780 2003-02-28
As used herein, "codon-optimized nucleic acid sequence" or "codon-optimized
polynucleotide"
or "synthetic ORF" or "synORF" refers to a codon-optimized nucleotide sequence
encoding a
PRRSV ORF protein or fragment thereof.
As used herein, "immune response" refers to a cellular or humoral response of
the immune
system.
As used herein, "specific immune response" refers to an adaptive immune
response, i.e. one that
is specific for an infecting pathogen, such as the production of antibodies
against a particular
pathogen. It may be naturally-induced, for example by a naturally infecting
pathogen, or
artificially induced, for example by passive or active immunization. A
specific immune response
is distinct from a non-specific or innate immune response. A non-specific or
innate immune
response is not specific to a particular infecting pathogen, for example, the
response of
macrophages that are immediately available to combat a wide range of pathogens
without
requiring prior exposure. Non-specific modulators of the immune system are
modulators of the
non-specific immune repsonses. These concepts and distinctions are understood
in the art (see
for example Chapter 1 in " Immunobiology" 5th ed. CA Janeway et al. Garland
Publishing,
c2001 ).
As used herein, "immunologically active" or "biologically active" refers to an
activity that
promotes the generation of a specific immune response against PRRSV in an
animal, or in an in
vitro, ex vivo or other system that is predictive of a specific immune
response against PRRSV in
an animal. This activity can be the result of a direct interaction with the
immune system or
isolated components of the immune system, such as the activity of an antigenic
peptide fragment
binding an antibody. A molecule, such as a peptide, is also considered
immunologically active
when it promotes the generation of an anti-PRRSV immune response that does not
involve a
direct interaction with the immune system or components thereof. Examples of
interaction that
indirectly generate an anti-PRRSV immune response include stabilization of an
antigenic
conformation of an antigenic peptide, when the stabilizing component does not
itself necessarily
have antibody-binding epitopes. A peptide that is not antigenic in the absence
of other peptides
is nonetheless considered immunotogically active if it participates directly
in the generation of an
antigenic epitope upon homo- or hetero- dimerization. In the context of this
invention,
"immunologically active" does not refer to activities that non-specifically
modulate the immune
system, i.e activities that modulate a non-specific or innate immune response.
"Stringent" hybridization conditions are those that (1) employ low ionic
strength and high
temperature for washing, for example, 0.015 M NaCI/0.0015 M sodium citrate /
0.1%SDS at
14
CA 02418780 2003-02-28
50C), or (2) employ during hybridization a denaturing agent such as formamide,
for example,
50% (vol/vol) formamide with 0.1% bovine serum albumin/0.1%Ficoll/0.1%
polyvinylpyrrolidone/50 nm sodium phosphate buffer at pH 6.5 with 750 mM NaCI,
75 mM
sodium citrate at 42C. Another example is hybridization in SO% formamide, 5x
SSC (0.75M
NaCI, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium
pyrophosphate,
5x Denhardt's solution, sonicated salmon sperm DNA (50 ug/ml), 0.1% SDS, and
10% dextran
sulfate at 42C, with washes at 42C in 0.2x SSC and 0.1% SDS. A skilled artisan
can readily
determine and vary the stringency conditions appropriately to obtain a clear
and detectable
hybridization signal.
Codon-optimization of PRRSV ORF nucleic acid sequences
Preferential codon usage is known in the art and preferred codons for
expression in mammalian
cells are shown in Table 2. Codon optimization has been reported with varying
degrees of
success. See, for example, Grosjean et al. (1982) Gene 18:199-209, Haas et al.
(1998) Current
Biology 6:315-324, Andre et al. (1998) J. Virol. 72:1497-1503, Corbet et al.
(2000) AIDS Res
Hum Retroviruses 16:1997-2008. Codon optimization is described in W097/28273,
W099/03997, WO00/65076 and WO00/188141.
The genomic sequences for a number of PRRSV strains are known in the art, and
many have
been deposited in GenBank, the NIH genetic sequence database. Polynucleotide
sequences
encoding proteins of North American PRRSV strains are used in the instant
application.
Examples of PRRSV strains and accession numbers for their genomic and/or
protein-encoding
sequences are given in Table 1. The wild-type ORF sequences for PRRSV strains
not listed in
GenBank are readily obtainable by those skilled in the art by generally
applicable molecular
biology techniques such as Southern blotting and colony hybridization.
PRRSV ORFs contemplated for use in the present invention include ORFS, ORF6,
ORF4,
ORF2a, or ORF2b, ORFIa, ORFIb, ORF3 or ORF7, in which case they could be
referred to, for
example, as synORFS, synORF6, synORF4 or synORF2a, or synORF2b, synORFla,
synORFlb,
synORF3 or synORF7 respectively.
wtORF sequence GenBank Reference
(IAF-Klop strain of PRRSV) I Accession No.
ORF2 AF003343 Gonin et al.,
ORF3 AF003344 I (1999) J Vet Diagn Invest 11:20-26
ORF4 AF003345 ~
ORES U64928 j Pirzadeh et al.,
CA 02418780 2003-02-28
ORF6 U64928 (1998) Can J Vet Res 62:170-177
ORF7 U64928 Gagnon et Dea,
(1998) Can J Vet Res 62:110-116
In an exemplary embodiment, the unoptimized ORF nucleic acid sequences are
from IAF-BAJ
(e.g. GenBank accession # U64929), or from IAF-94-287 (e.g. U64934), or from
the Quebec
reference cytopathic strain IAF-Klop (Mardassi et al., (1994) Can. J. Vet.
Res. 58:55-64;
Mardassi et al. , (1995) Arch. Virol. 140:1405-1418), e.g. GenBank Accession
Number U64928.
The unoptimized PRRSV nucleic acid sequence upon which the optimized synORF
sequence is
based can encode a wild type PRRSV polypeptide or a modification (mutation) of
the wild-type
PRRSV amino acid sequence. Illustrative amino acid modifications are encoded
by nucleotide
substitutions, additions, deletions, inversions and transversions. The
modified PRRSV
polypeptides encoded by the colon-optimized sequences of the invention are
biologically active
(immunologically active). In accordance with the present invention, the
contemplated amino
acid sequence modifications include, but are not limited to, those that
contribute to the utility of
the synORF polynucleotide sequences of the invention, for example as a
vaccine, by improving
efficacy (immunological activity) and/or bioavailability of the synORF
expression product
and/or reduce its toxicity. Such modifications may give rise to mutant
po(ypeptides that are
functionally altered with respect to, for example, transcription or
translation efficiency, sequence
length (e.g. truncated polypeptides), intracellular location, interactions
with other polypeptides
(e.g. homo- or hetero- dimerization), and/or undergo altered post-
translational processing of the
polypeptide, for example by mutation of phosphorylation sites, or asparagine
residues that are N-
glycosylation sites. Alternatively, synORF potynucleotide sequences of the
invention can be
based on unoptimized PRRSV polypeptide sequences that incorporate
modifications introduced
for technical reasons, e.g. for technical convenience.
In accordance with the present invention, an optimized ORF nucleic acid
sequence can be a
completely optimized sequence in which all of the non-preferred colons are
replaced by
preferred colons, or a partially optimized nucleic acid sequence in which less
than all of the non-
preferred colons are replaced by a preferred colon. For example, I or more, 2
or more, 3 or
more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or
more, 11 or more,
12 or more, 15 or more, 20 or more, or 30 or more or 40 or more or 50 or more,
or 75 or more or
100 or more, non-preferred colons may be replaced by preferred colons, or at
least about 50%,
60%, 70%, 80%, 90%, 95%, or at least about 98% of the non-preferred colons may
be replaced
by preferred colons, or all but 10, all but 9, all but 8, all but 7, all but
6, alt but 5, all but 4, all
16
CA 02418780 2003-02-28
but 3, all but 2, or all but 1 of the non-preferred codons are replaced by
preferred codons. In
other words, the percent of non-preferred codons replaced by preferred codons
may be 100% or
less, 98% or less, 95% or less, 93% or less, 90% or less, 85% or less, 80% or
less, 75% or less,
66% or less, 60% or less, 50% or less, 40% or less, 30% or less, 20% or less,
or 10% or less, for
example.
Candidate synthetic sequences are tested to determine if the complete or
partial optimization has
improved expression over that of the unopt.imized ORF polynucleotide sequence.
Efficacy of the
construct in eliciting an immune response and cytotoxicity of the constructs
can also be assessed,
according to the methods described below (see also Examples).
In accordance with the present invention, a synORFS sequence based on the IAF-
Klop strain is
provided, having 130 nucleotide substitutions such that all of the non-
preferred codons of the
unoptimized sequence are replaced with preferred codons in the optimized
synORFS
polynucleotide (see Figure 2, SEQ ID NO: 1). A related embodiment of the
invention is a
synthetic ORFS polynucleotide variant ("synORFS variant") designed from a
modified ORFS
sequence and having less than all codons optimized (see Figure 8, SEQ ID NO:
40). The
nucleotide sequence of a third codon-optimized ORFS sequence, "synORFS
variant2", is
provided in SEQ ID NO: 71 and encodes the wild type amino acid sequence of
ORFS of IAF-
Klop (and thus the same polypeptide as synORFS). The synORFS variant2 and
synORFS
nucleotide sequences differ in 6 positions.
Completely optimized synORF4 and synORF6 polynucleotide sequences are also
provided (see
Figure 10 and Figure I 1 ) and, in accordance with the present invention,
synORF sequences can
also be based on the unoptimized sequences of ORFIa, ORFlb, ORF2a, ORF2b, ORF3
or
ORF7.
The codon-optimized ORF nucleic acid sequences according to the present
invention can encode
an entire nucleic acid sequence of a PRRSV ORF polynucleotide or one or more
fragments
thereof that are biologically active (immunologically active). Fragments of
ORF nucleic acid
molecules typically encode ORF protein fragments that are themselves antigenic
or that can less
directly enhance an anti-PRRSV immune response (in vitro and/or in vivo). Such
fragments will
generally contain at least 7, or at least 10 amino acids to generate an
epitope, and typically these
will be consecutive amino acids from the PRRSV polypeptide sequence.
Candidate fragments are selected non-randomly, for example based on evidence
predicting that
they will be antigenic or less toxic, e.g. based on hydrophilicity plots
(Figure I2) or experimental
17
CA 02418780 2003-02-28
data. For example work by Fernandez et al. ((2002) Virus Res 83:103-118)
indicates that
domains within the N-terminal 119 amino acids of PRRSV ORF5 play a major rote
in the
induction of apoptosis, and thus of cytotoxicity that might lower expression
of synORFS
polynucleotides of the invention. Alternatively or in addition, fragments can
be randomly
selected and screened for immunological activity. The fragments can be
screened for
immunological activity using in vitro and/or in vivo methods described below
(see also the
Examples). In accordance with the present invention candidate fragments can
also be screened
for bioavailability (e.g. expression) and/or toxicity and/or other
characteristics considered
relevant to the uses of the present invention by a worker skilled in the art
(see below, and
Examples).
Antigenic peptides that can be used include, for example, fragments that
contain one or more
neutralizing epitopes, including fragments that form the extracellular
domains) of the PRRSV
polypeptide. In an exemplary embodiment, such a fragment will be from the
hydrophilic region
of an ORFS polypeptide, for example nucleotides 106-156 (amino acids 36-52) of
an ORFS
protein of the IAF-Klop strain (Plagemann et al., (2002) Arch Virol 147:2327-
2347), or smaller
or larger fragments containing consecutive sequences of at least 7 or at least
10 amino acids from
within this domain. SynORF polynucleotides of the invention can also encode
ORFS
polypeptide fragments such as amino acids 1-42 (nucleotides I-126), and/or
amino acids 47-200
(nucleotides 139-603); in another illustrative embodiment these synORFS
polynucleotides are
encoding peptide fragments from a modified ORFS protein of IAF-94-287 PRRSV
strain.
Methods of producing fragments for activity screening purposes include, but
are not limited to
cleavage of precursor molecules (for example using restriction enzymes for DNA
molecules and
proteases for polypeptides)> and chemical, enzymatic or other synthetic
methods known in the
art. Sequences encoding the synthetic polynucleotide fragments for use in the
instant application
will typically be synthesized using molecular biological methods.
In accordance with the present invention a codon-optimized ORF nucleic acid
molecule can
comprise one or more ORF genes, one or more ORF gene fragments, or a
combination thereof.
For example, without necessarily including the entire ORF sequence, one, more
than one, or all
of the epitopes of an unoptimized protein can be encoded on same codon-
optimized ORF nucleic
acid molecule and used, for example, to immunize swine against PRRSV. As there
are common
PRRSV antigenic epitopes among different PRRSV strains, it is contemplated
that synORFs
designed based on one strain of PRRSV could be effective to immunize swine
against other
strains of PRRSV. It is also envisaged that the synthetic codon-optimized ORF
sequence of the
18
CA 02418780 2003-02-28
present invention can encode epitopes from more than one strain of PRRSV,
thereby providing
for immunization against more than one strain of PRRSV.
The synORF sequences of the invention may be chosen for their ability to evoke
an immune
response, in vitro or in vivo, when provided alone or when provided in
combination with another
S molecule. For example, an immune response that is elicited in a pig by a
polypeptide may be
enhanced by the presence of another polypeptide that does not by itself evoke
an immune
response. For example, a peptide may indirectly contribute to an immune
response by binding
with another molecule such that it is processed more favourably, is
stabilized, is less cytotoxic,
or shares one or more new epitopes are generated that are useful in the
immunization of swine
against PRRSV. The mechanism by which a peptide might indirectly enhance an
immune
response is not critical to the use of the sequence in instant invention, and
in the context the
peptide is considered biologically active in the context of immunization. In
accordance with the
present invention, such peptides may be provided as codon-optimized PRRSV ORF
polynucleotide sequences or fragments thereof.
I S Selection of sequences
Sequences for use in the instant invention, including complete PRRSV ORF
sequences and
fragments, wild-type or modified, may be selected based on their known or
predicted beneficial
biological activities and/or lack of undesirable effects. Alternatively or in
addition, candidate
fragments may be sampled from a library of fragments randomly generated, for
example, from a
particular PRRSV ORF or an entire PRRSV genome, and tested for expression,
efficacy and
toxicity. Candidate fragments may be tested preliminarily as unoptimized
sequences in addition
to being tested after codon-optimization. In an exemplary embodiment, an
adenovirus-based
library of truncated synORFS clones is constructed and truncated ORFS (GPs)
peptides expressed
and tested.
The ability to elicit a favourable immune response may be tested using a
variety of assays known
in the art. For example a candidate sequence may be administered to a suitable
animal such as a
pig in an in vivo challenge, and at one or more appropriate times post-
challenge the animal
serum collected and assayed for the presence of specific antibodies by any of
the various suitable
serological test procedures known in the art. Exemplary assays include virus
neutralization,
indirect immunofluorescence (IIF), ELISA, blastogenic transformation test,
virus isolation,
Western Blot, necropsy and histopathological examination (see below). Animals
other than the
pig, such as mice, may also be useful for such in vivo assays. A worker
skilled in the art will be
19
CA 02418780 2003-02-28
able to determine which animals are suited to such uses, based on parameters
such as the
expression vector used, for example.
In vitro assays may alternatively or additionally be used to assess efficacy
of candidate
sequences. For example, cell lines expressing peptides from an expression
vector of the instant
invention may be tested for immunoreactivity with available polyclonal or
monoclonal
antibodies that are known to bind epitopes of the full-length ORF sequence
from which the test
sequence was derived.
It is desirable to accomplish the immunization of swine against PRRSV with
minimal side-
effects. Cytotoxicity related to the expressed sequence may be reduced or
avoided by selection
of appropriate expression sequences. For example, PRRSV ORFs sequences for use
in the
instant invention may be included or excluded based on their ability to cause
or prevent
apoptosis. To test for toxicity in vitro, for, example, cells may be infected
with adenoviral
vectors expressing a candidate sequence and monitored, at various times post-
infection, using a
number of assays. A worker skilled in the art will be able to select an assay
appropriate for the
detection of cytotoxicity. As indicators of apoptosis, abnormal proliferation
or gross cellular
changes may be observed visually. Other exemplary assays of apoptosis include
DNA
fragmentation assays (e.g. using TUNEL microscopy), and caspase activity
assays. Suitable cells
for use in such assays include MARC-145 cells and alveolar macrophages. (See
below).
Generation of codon-optimized PRRSV ORF nucleic acid molecules
Replacement of non-preferred codons in the unoptimized ORF nucleic acid
sequence with
preferred codons to generate a synORF sequence can be achieved using standard
techniques
known in the art. For example, the optimized nucleic acid sequence may be
chemically
synthesized in its entirety in vitro, or fragments of the unoptimized sequence
may be replaced by
chemically synthesized polynucleotides containing the requisite nucleotide
changes.
Alternatively, the optimized nucleic acid sequence can be assembled using one
or more
amplification reactions, such as PCR. Methods of generating synthetic genes in
this manner are
known in the art. For example, in one embodiment, synthetic oligonucteotides
designed to cover
the entire ORF gene and contain suitable nucleotide overlaps, can be
synthesized and used in a
series of single overlap polymerise chain reactions to reconstruct a synthetic
(codon-optimized)
form of the full gene (Holler et al. , (1993) Gene 136:323-328). Optionally,
following the first
single overlap PCR, the amplified DNA product is isolated before use in a
subsequent single
overlap PCR amplification with new primers. This procedure is repeated until
the entire codon-
optimized gene had been assembled.
CA 02418780 2003-02-28
Cloning codon-optimized PRRSV ORF nucleic acid molecules into an expression
vector
A codon-optimized ORF nucleic acid sequence can be designed and constructed as
part of an
expression cassette, and may be combined with elements that direct efficient
transcription and
translation of the inserted DNA. Desirable elements include such components as
promoters,
enhancers, polyadenylation signals and terminators. Such elements can integral
to the expression
cassette containing a codon-optimized ORF nucleic acid sequence, and/or
present in an
expression vector.
In one embodiment of the present invention, the hCMV promoter is used as it
has previously
been found to be very efficient in DNA immunization experiments in pigs
against Aujesky's
disease (Gerdts et al., (1997) J Gen Virol. 78( Pt 9):2139-46) and PRRSV
(Pirzadeh and Dea,
(1998) J. Gen. Virol. 79:989-999. The hCMV provides for constitutive
expression of the
downstream gene. The use of other promoters, such as other CMV promoters or
tissue specific
promoters is also contemplated.
In an exemplary embodiment, a regulatable promoter such as the CMV cumate
promoter or a
tetracycline derivative responsive promoter, may be preferable if control over
expression of the
downstream gene is desirable, for example if constitutive expression would
cause cellular
cytotoxicity (Massie et al., (1998) Cytotechnolo~y 28:53-64). In another
embodiment of the
present invention the ORF gene is placed downstream of a tetracycline
regulated TR5 promoter
to permit controlled expression of the gene. A tTA or rtTA regulation system
might alternatively
be used, allowing the control of expression of the transgene either by
administering tetracycline
or withdrawing its administration respectively. An exemplary terminator
element for use in the
practice of the invention is the bovine growth hormone terminator. It would
also be apparent to
one of skill in the art that other regulatable promoters, and terminators
could be used in the
expression cassettes of the expression vectors of the invention.
It is contemplated that co-expression of one or more other proteins may be
desirable in the
practice of the invention, and to this end the expression cassettes and
vectors of the invention can
be adapted for co-expression of a plurality of polynucleotides, at least one
of which would be a
synORF polynucleotide of the invention. The expression cassette containing the
synORF
polynucleotide of the invention can include the polynucleotide sequence
encoding another
peptide positioned directly before or after the synORF polynucleotide, so as
to generate a fusion
protein upon expression. Peptides useful in such fusion proteins include but
are not limited to
peptides that serve as haptens to enhance immune response, and marker proteins
such as
21
CA 02418780 2003-02-28
fluorescent polypeptides to facilitate expression monitoring, e.g. Green
Fluorescent Protein
(GFP).
Alternatively, a synORF expression cassette can be a dicistronic or
polycistronic expression
cassette, encoding at least one other polynucleotide sequence that is
transcribed and translated to
generate a distinct peptide product, co-expressed with the synORF
polynucleotide. An IRES
(internal ribosomal entry site) sequence can be inserted between
polynucleotide sequences to
allow continuous transcription of the two or more polynucleotide sequences
starting from a
promoter upstream of the most 5' sequence. Alternatively transcription for
different co-
expressed sequences can be initiated from different promoters.
In another approach, a synORF polynucleotide and one or more polynucleotides
for co-
expression with the synORF polynucleotide can be placed into separate
expression cassettes
and/or into separate expression vectors. Co-expression of non-fusion proteins
in any of the
above-described expression cassette and expression vector combinations can be
arranged, if
desired, for example by using the same regulatable promoter system to direct
transcription of
synORF and other polynucleotide(s).
Peptides that may be co-expressed with a codon-optimized ORF nucleic acid
sequence of the
invention include other PRRSV ORFs and other immunologically active proteins
or fragments
thereof, and cytokines and other non-specific modulators the immune system or
fragments
thereof. Exemplary cytokines include gamma-interferon, IL-2, IL-4 (Hornef et
al., (2000) Med
Microbiol Immunol 189:97-104), IL-12 (Palendira et al., (2002) Infect Immun
70:1949-1956),
IL-5, IL-6 (Braciak et al., (2000) Immunology 101:388-396).
Experimental evidence indicates that an interaction between protein M (encoded
by ORF6) and
GP5 (encoded by ORFS) of a PRRSV-related virus enhances the immune response
when co-
expressed (Balasuriya et al., (2000) J Virol 74:10623-10630; Balasuriya et
al., (2002) Vaccine
20:1609-1617), though a response was not seen with ORFS alone. In an exemplary
embodiment,
synORFS or fragments thereof might be co-expressed with a fragment or complete
PRRSV
peptide such as ORF6 or ORF2a or ORF4. Other examples include PRRSV ORFla,
ORFlb,
ORF2b, ORF3 or ORF7. The polynucleotides encoding these ORFs, or fragments
thereof, may
be wild-type or modified sequences, unoptimized sequences, or optimized
sequences of the
invention.
Once the expression cassette containing the codon-optimized ORF nucleic acid
sequence has
been constructed, it may be inserted into an appropriate expression vector.
Such expression
vectors are well known in the art and include bacterial plasmids, modified
viral nucleotides (e.g.
22
CA 02418780 2003-02-28
retrovirus, vaccinia virus, baculovirus, adeno-associated virus, poxvirus or
adenovirus), phage
DNA, and combinations of plamid and phage or viral DNA (e.g. phagemids). An
appropriate
expression vector is chosen based on a number of parameters well known in the
art, including for
example, host cell, expression control, expression efficiency and technical
feasibility.
I. Plasmids
In one embodiment of the invention, the codon-optimized ORF nucleic acid
sequences are
inserted into a plasmid vector. Plasmid vectors offer many advantages.
Firstly, methods of
generating and purifying plasmid DNA are rapid and straightforward. This fact,
combined with
simple quality control, facilitates technology transfer and reduces the cost
of production.
Secondly, plasmid DNA typically does not integrate into the genome of the host
cell, but is
maintained in an episomal location as a discrete entity eliminating
genotoxicity issues that
chromosomal integration may raise.
A variety of plasmids are now readily available commercially and include those
derived from
Escherichia coli and Bacillus subtilis, with many being designed particularly
for use in
mammalian systems. Specific plasmids that could be used in the present
invention include, but
are not limited to, the eukaryotic expression vectors pRc/CMV (Invitrogen),
pCR2.1
(Invitrogen), pAd/CMV and pAd/TRS/GFPq (Massie et al., (1998) Cytotechnology
28:53-64).
In an exemplary embodiment, the ptasmid is pRc/CMV, pRc/CMV2 (Invitrogen),
pAdCMVS
(IRB-NRC), pcDNA3 (Invitrogen), pAdMLPS (IRB-NRC), or pVAX (Invitrogen).
In one embodiment, the plasmid contains a codon-optimised ORFS nucleic acid.
In a related
embodiment, the plasmid is pRc/CMV/synORFS, pAd/TRS/GFPq/synORFS,
pRc/CMV2/synORFS, pAdCMVS/synORFS, pcDNA3/synORFS, pAdMLPS/synORFS, or
pVAX/synORFS. In exemplary embodiments, the plasmid is pAd/TRS/GFPq/synORFS.
II. Viral vectors
In another embodiment of the present invention, the codon-optimized ORF
nucleic acid sequence
is inserted into a virus or engineered construct derived from a viral genome.
The ability of certain
viruses to enter cells via receptor-mediated endocytosis and to integrate into
host cell genome
and express viral genes stably and efficiently have made them attractive
candidates for the
transfer of foreign genes into mammalian cells. A viral vector is often but
not necessarily
replication-defective. The use of such a replication-competent virus vector
that is packaging-
and/or dissemination- defective is also contemplated. In an illustrative
embodiment, a
dissemination-defective virus vector is capable of replication in infected
cells, but does not
23
CA 02418780 2003-02-28
encode the protein (e.g. adenovirus protease) necessary for viral particle
assembly and
dissemination so replication does not proceed beyond a first round. See, for
example, Elahi et al.
(Gene Ther (2002) 9:1238-1246 and U.S. Patent No. 6,291, 266). The nature of
the viral vector
is not otherwise believed to be crucial to the successful practice of the
invention.
In one embodiment of the present invention, the codon-optimized ORF nucleic
acid sequence is
inserted into an adenovirus vector. An advantage of adenovirus vectors is
that, like plasmid
DNA, they typically do not integrate into the host genome and thus foreign
genes delivered by
adenovirus vectors remain episomal (Graham and Prevec (1991) Meth. Mot. Biol.
7:109-128).
The human adenovirus (hAdV) may be one of the 47 or more different known
serotypes or
subgroups A-F, hAdV type 2 and hAdV type 5 (hAdVS) are often used. Generation
and
propagation of replication-defective human adenovirus vectors requires a
unique helper cell line.
Helper cell lines may be derived from human cells such as human embryonic
kidney cells,
muscle cells, hematopoietic cells or other human embryonic mesenchymal or
epithelial cells.
Alternatively, the helper cells may be derived from the cells of other
mammalian species that are
permissive for human adenovirus, i.e. that provide, in trans, a sequence
necessary to allow for
replication of a replication-deficient virus. Such cells include, for example,
293 cells, Vero cells
or other monkey embryonic mesenchymal or epithelial cells. The use of non-
human adenovirus
vectors, such as porcine or bovine adenovirus vectors is also contemplated. A
worker skilled in
the art will be able to select an appropriate viral vector and helper cell
line if necessary.
In one embodiment of the present invention, expression of a gene of the
present invention can be
accomplished, for example, using the Ad/CMVIacZ expression vector, a
replication-defective
E1-deleted (and, optionally, E3-deleted) hAdVS, propagated in 293 helper cells
(ATCC CRL-
1573) to complement the functions of the E1-deleted genomic region of
Ad/CMV/gene and
thereby permit the replication of replication-defective hAdVs.
In an exemplary embodiment of the present invention, the adenovirus "shuttle"
or "transfer"
vector is pAd/TR5 or pAd/TR5/GFPq (Massie et al., (1998) Cytotechnology 28:53-
64), derived
from the adenovirus type 5 of subgroup C, and the helper cell line is 293,
derived from human
embryonic kidney cells (Graham et al., (1997) J. Gen. Virol. 36 :59-72). In
this shuttle vector,
the tetracycline-regulable promoter, TRS, drives expression of the gene. The
expression cassette
replaces the E1 gene and is flanked on one end by the encapsidation and
packaging signals and
on the other end by an adenovirus sequence allowing recombination and
generation of
replication-defective recombinant virus. In this cassette, which was derived
from pAdBMS (US
Patent No. 5, _518,913), expression of heterologous genes is optimized by the
presence of the
24
CA 02418780 2003-02-28
adenovirus tripartite leader sequence and the adenovirus major late enhancer
flanked by splice
donor and acceptor sites.
In this exemplary system, recombinant adenovirus is generated by homologous
recombination
between the shuttle vector and a provirus vector. Due to the possibility of
recombination between
two proviral vectors, wild-type adenovirus may be generated from this process;
therefore, it is
customary to isolate a single clone of the virus from an individual plaque and
examine its
genomic structure, if wild-type (dissemination-competent) viruses are to be
avoided, as is usually
the case. The use of a two plasmid based approach, or of the YAC system or
other alternative
approaches known in the art for the production of recombinant adenovirus are
also contemplated.
III. Construction of an expression vector
Those skilled in the field of molecular biology will understand that a variety
of routine methods
may be employed to clone the codon-optimized ORF gene (and control non-
optimized gene) or
gene fragments into the required vectors and that the expression construct may
then be replicated
in and isolated from a number of possible host organisms.
In one embodiment of the present invention, once a synORF gene or fragment
thereof has been
constructed, the entire synORF gene is amplified by PCR using a forward primer
that comprises
the first ATG codon of the synORF gene downstream of a Kozak motif for
initiation of
translation in vertebrates (Kozak (1987) Molecular Biology 196:947-950), and a
reverse primer
that comprises the stop codon of the viral gene.
Alternatively, for directional cloning, restriction sites can be added at the
5' ends of the sense and
antisense primers respectively (Pirzadeh and Dea (1987) J. Gen. Virol. 79:989-
999), and the
synORF gene, or fragment thereof, can then be cloned into the corresponding
restriction sites of
an expression vector, downstream of the vector promoter, producing a
recombinant plasmid
useful for the expression of the synthetic codon-optimized ORF gene product.
For construction of recombinant viral vectors, the synORF nucleic acid
sequence or expression
cassette is typically inserted into a shuttle vector and recombinant viral
vectors are subsequently
generated by co-transfecting the helper cell line with the shuttle vector and
simultaneously
infecting them with the helper virus. In another embodiment of the present
invention, a PCR
amplified synORF gene is inserted into a unique restriction site of an
adenovirus shuttle vector,
in which expression of the synORF gene is under the control of a regulatable
promoter.
Recombinant adenoviruses are subsequently generated in helper cells by
homologous
recombination with a replication defective vector such as Ad/CMVIacZ, as
detailed in Jani et al.
CA 02418780 2003-02-28
(1997) J. Virological Methods 64:111-124. Recombinant viruses are identified
by analysing for
expression of the protein encoded by the ORF nucleic acids. For example,
recombinant
Ad/TR5/GFPq/synORFS viruses are identified by analysing for expression of the
recombinant
GP5 protein, for example by Western immunoblot analysis or
radioirnmunoprecipitation assays.
Delivery of codon-optimized PRRSV ORF nucleic acid molecules into mammalian
cells
In order to effect expression of ORF constructs, the expression construct must
be delivered into a
cell. This delivery can be accomplished in vitro, as in laboratory procedures
for transfecting or
transforming cells lines, in vivo, or ex vivo (see below). As described above,
an exemplary
mechanism for delivery is via viral infection where the expression construct
is encapsidated in an
infectious viral particle.
In accordance with the present invention, recombinant viruses such as
adenoviruses can be
administered to different animal tissues via a variety of routes including,
for example,
subcutaneous and intraperitoneal administration, trachea instillation (
Rosenfeld etal., (1992)
Cell 68:143-155), muscle injection (Ragot et al., (1993) Nature 361:647-650),
peripheral
intravenous injection (Herz and Gerard (1993) Proc. Natl. Acad. Sci. USA
90:2812-2816), and
stereotactic inoculation into the brain (Le Gal La Salle et al., (1993)
Science 259:988-990).
Several non-viral methods for the transfer of the synORF nucleic acid
molecules, expression
cassettes, expression vectors into cultured mammalian cells also are
contemplated by the present
invention. These include calcium phosphate co-precipitation technique, linear
25 kDa
polyethyleneimine (PEI), DEAF-dextran, electroporation, direct microinjection,
DNA-loaded
liposomes, lipofectamine-DNA complexes, cell sonication, gene bombardment
using high
velocity microprojectiles> and receptor-mediated transfection, all of which
are known in the art.
The use of "naked" DNA to deliver DNA to cells is also known in the art
(Felgner). Some of
these techniques may be successfully adapted for in vivo or ex vivo use.
In an exemplary embodiment, non-viral constructs would be delivered to swine
via intradermal
and/or intramuscular injection using a gene gun transfection system (see
below) or using a 27-
gauge needle The intramuscular injection is given into the tibialis cranalis
muscle, whereas
the intradermal injection is given into the dorsal surface of the ear or in
the skin beneath the ear.
Transferring DNA expression constructs into cells could involve particle
bombardment (Johnston
30 and Tang (1994) "Gene Gun Transfection of Animal Cells and Genetic
Immunization" In
Methods in Cell Biology, 43:353-366). This method depends on the ability to
accelerate DNA
coated microprojectiles to a high velocity allowing them to pierce cell
membranes and enter cells
without killing them. Several devices for accelerating small particles have
been developed. One
26
CA 02418780 2003-02-28
such device relies on a high voltage discharge to generate an electrical
current, which in turn
provides the motive force. The microprojectiles used have consisted of
biologically inert
substances such as tungsten or gold beads. Selected organs, including the
liver, skin, and muscle
tissue of rats and mice, have been bombarded in vivo. This may require
surgical exposure of the
S tissue or cells, to eliminate intervening tissue between the gun and the
target organ, i.e., ex vivo
treatment. Again, DNA encoding an ORF may be delivered via this method within
the scope of
the present invention.
In a further embodiment of the invention, the expression construct may be
entrapped in a
liposome. Liposomes are vesicular structures characterized by a phospholipid
bilayer membrane
and an inner aqueous medium. Multilamellar liposomes have multiple lipid
layers separated by
aqueous medium. They form spontaneously when phospholipids are suspended in an
excess of
aqueous solution. The lipid components undergo self-rearrangement before the
formation of
closed structures and entrap water and dissolved solutes between the lipid
bilayers. Also
contemplated are lipofectamine-DNA complexes. Liposome-mediated nucleic acid
delivery and
expression of foreign DNA in vitro has been very successful in cultured chick
embryo, HeLa and
hepatoma cells. Nicolau et al., ((1987) Methods in Enzynolo~,y 149:157-176)
accomplished
successful liposome-mediated gene transfer in rats after intravenous
injection.
In certain embodiments, gene transfer may more easily be performed under ex
vivo conditions.
Ex vivo gene therapy refers to the isolation of cells from an animal, the
delivery of a nucleic acid
into the cells in vitro, and then the return of the modified cells back into
the animal. This may
involve the surgical removal of tissue or organs from an animal or the primary
culture of cells
and tissues. U.S. Pat. No. 5,399,346 discloses ex vivo therapeutic methods.
In one embodiment of the invention, the expression construct may consist of
DNA plasmids.
Transfer of the construct may be performed, for example, by one of the methods
mentioned
above that physically or chemically permeabilizes the cell membrane. This is
particularly
applicable for transfer in vitro, but it may be applied to in vivo use as
well. Dubensky et al.
(1984) Proc. Natl. Acad. Sci. USA 81:7529-7533 successfully injected
polyomavirus DNA in the
form of CaP04 precipitates into liver and spleen of adult and newborn mice
demonstrating active
viral replication and acute infection. Benvenisty and Neshif (1986) Proc.
Natl. Acad. Sci. USA
83:9551-9555 also demonstrated that direct intraperitoneal injection of CaP04
precipitated
plasmids results in expression of the transfected genes. It is envisioned that
ORF DNA could also
be transferred in vivo in a similar manner to express the encoded protein.
27
CA 02418780 2003-02-28
Once the expression construct has been delivered into the cell, the ORF
nucleic acid can locate to
a variety of intracellular sites, for example it can be stably maintained in
the cell as a separate,
episomal segment of DNA. Such nucleic acid segments or "episomes" typically
encode
sequences sufficient to permit maintenance and replication independent of or
in synchronization
S with the host cell cycle. How the expression construct is delivered to a
cell and where in the cell
the nucleic acid remains is dependent on the type of expression construct
employed.
Primary mammalian cell cultures may be prepared in various ways. In order for
the cells to be
kept viable while in vitro and in contact with the expression construct, it is
necessary to ensure
that the cells maintain contact with the correct ratio of oxygen and carbon
dioxide and nutrients
but are protected from microbial contamination. Cell culture techniques are
well documented.
During in vitro culture, the expression construct may deliver and express
protein encoded by an
ORF into the cells. The cells may then be reintroduced into the original
animal, or administered
into a different animal, in a pharmaceutically acceptable form, for example by
one of the means
described below.
Evaluation of expression of the codon-optimized ORF construct
Verification of the in vivo expression of PRRSV protein from an ORF expression
vector by
immunizing CD-1 or BALBlc mice.
The in vivo expression of a PRRSV protein from a vector, for example the
expression of GPS
from the pRc/CMVS plasmid vector, can be shown following genetic immunization
of CD-1 and
Balb/c mice. See, for example, Pirzadeh and Dea, (1998), J. Gen. Virol. 79:989-
999.
Indirect Immunofluorescence (IIF) assay for transient expression
Transient expression experiments in confluent monolayers of 293 cells may be
used to assay for
the expression of a pRc/CMV/synORF construct. Cells can be easily transfected
with the plasmid
by use of the Fugene 6 Transfection ReagentT"~' (Roche Diagnostics) and
expression of the ORF
protein product can be visualized by indirect immunofluorescence (IIF). In
this procedure, cells
are fixed at various times post-transfection, then reacted with anti-ORF
rabbit monospecific
hyperimmune serum (Mardassi et al., (1996) Virology 221:98-I 12) and the
immune reaction
revealed following incubation with fluorescein-conjugated goat anti-rabbit Ig
(Boehringer
Mannheim) as previously described (Loemba et al., (1996) Archives of Virology
141:751-761).
Fluorescein fluorescence is indicative of the presence of the PRRSV protein
and consequently of
ORF expression.
28
CA 02418780 2003-02-28
Direct fluorescence from a Reporter Protein
To easily monitor expression of the recombinant clones or hAdVs, a gene
encoding for a
fluorescent protein such as the GFPq protein (Green fluorescent protein) can
be used as a reporter
gene. For example, the reporter protein can be cloned downstream of a multiple
cloning site in
an expression vector, e.g. an adenoviral shuttle vector, under the control of
an internal ribosomal
entry site (IRES) such that it is co-expressed with the upstream recombinant
clone. At various
times post-infection, the intensity of the fluorescence is observed as an
indication of recombinant
protein expression, i.e. fluorescence increases with an increase in ORF
protein expression.
Western immunoblot analysis
Alternatively, the expression of an ORF protein product in cells transfected
with an expression
construct can be tested by Western blot. Lysates of cells carrying the
expression construct are
subjected to sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-
PAGE) and
electrotransferred onto nitrocellulose membranes. The PRRSV protein can then
be visualized
following incubation of the membranes with an anti-ORF rabbit monospecific
hyperimmune
serum as described previously (Mardassi et al., (1996) Virology 221:98-112).
Radioimmunoprecipitation assay
Expression of an ORF polypeptide or fragment thereof may also be detected by
radioimmunoprecipitation assays (RIPA). Radiolabelling of the recombinant
protein produced in
transfected cells with (35S]-methionine can be carried out as described
previously (Mardassi et
al., (1996) Virology 221:98-112). Clarified lysates of the transfected cells
can then be reacted
with an anti-ORF rabbit monospecific hyperimmune serum and the resultant
immune complexes
adsorbed onto protein A-sepharose beads (Pharmacia). Electrophoresis of the
dissolved beads on
SDS-polyacrylamide gels followed by fluorography and autoradiography will
reveal the presence
of the immune complexes and, consequently, of the PRRSV protein.
In vitro evaluation of cytotoxicity of codon-optimized ORF nucleic acid
molecules
To evaluate the cytotoxicity of hAdV-expressed recombinant PRRSV ORF proteins
or fragments
thereof, monolayers of non-helper cells, such as MARC-145 cells, are co-
infected with the
adenovirus recombinant vector, for example with hAdV/TR5/ORF, and hAdV/CMV/tTA
in the
presence or absence of doxycycline. The hAdV/CMV/tTA permits constitutive
expression of
tTA in infected non-helper cells using the constitutive human CMV immediate-
early
promoter/enhancer. The tTA is essential to the expression of the foreign gene
in hAdV-infected
29
CA 02418780 2003-02-28
cells, and doxycycline (1 pg/mI) is used to inhibit the expression of the
foreign gene in hAdV-
infected cells (Massie et al., (1998) Cytotechnology 28:53-64). Alternatively,
if transfected into
TetOn 293 cells, which constitutively express the reverse tetracycline
transactivator (rtTA), then
the hAdV/CMV/tTA vector is excluded and the expression of the PRRSV protein
from hAdV
vectors enhanced when cultivated in the presence of doxycycline (1 ftg/ml).
Experimental
controls typically include mock-infected MARC-14.5 cells, and cells that have
been infected with
either hAdV/CMV/tTA or an hAdV/vector carrying the unoptimized ORF alone.
Abnormal proliferation or gross cellular changes that occur upon intracellular
synthesis of the
recombinant PRRSV protein are visualised under a light microscope (Leyca,
Leitz), for example
under epifluorescent and phase contrast microscopy, at various times post-
infection. MARL-145
cells are not permissive to replication defective (E1 deleted) recombinant
hAdVs since they do
not complement their E1 gene functions, so cellular degeneration or
abnormalities observed can
be attributed to the toxicity of the expression of synORF (encoded PRRSV
protein, or fragments
thereof). Synthesis of the recombinant proteins can be followed in parallel by
western blotting.
Assessing the immune response to vaccination
Clinical observations:
Vaccinated and unvaccinated pigs may be observed and examined for the
development of clinical
signs of respiratory disease, beginning 1 to 3 days and continuing for several
weeks after virus
challenge. For example, some of the principal signs of respiratory disease
include a decrease in
growth and in feed consumption, persistent hyperthermia, eyelid oedema,
laboured breathing
(abdominal respiration) and/or rasping and crowing sounds heard during
inspiration.
Virus neutralization (VN) and serological tests
To test for the generation of an immune response to a vaccine, challenged
animals are typically
bled on day 0 (control) and on several occasions in the weeks and months post-
challenge. Long
term effects of vaccination can be determined by testing serum samples
collected months and
years post-challenge. The sera can be assayed for the presence of specific
antibodies by any of
the various suitable serological test procedures known in the art, for example
by virus
neutralization, indirect immunofluorescence (IIF), ELISA and Western Blotting.
In a virus neutralization (VN) test, also known as a seroneutralization assay,
serial dilutions of
heat-inactivated test and control serum samples are pre-incubated with a
constant dose of 100
CA 02418780 2003-02-28
TCIDS~ of the infective agent and then added to confluent layers of test
cells. The cells are
monitored regularly for cytopathic effects, and after about 5 days fixed and
tested for expression
of a viral protein other than that encoded by the vaccine. For example, if
ORFS is being used in
the vaccine, then expression of PPRSV nucleocapsid (N) protein can be
monitored by IIF using
the N protein specific monoclonal antibody Mab IAF-K8 (Dea et al. , (1996) J.
Clin. Microbiol.
34:1488-1493). Neutralizing titres are expressed as the reciprocal of the
highest dilution that
completely inhibits the expression of viral N protein.
A competitive ELISA for detection of antibodies to PRRSV using recombinant E.
coli-expressed
N protein as antigen can also be used to monitor humoral immune response
following
challenging of hyper-immunized pigs (Dea et al., (2000) J. Virol. Methods
87:109-122). A
commercial indirect ELISA (Idexx) for detection of anti-PRRSV antibodies can
also be used,
following the manufacturer's directions. Alternatively, or in addition, the
presence of anti-
PRRSV antibodies in the sera of the immunized animals can be assayed by other
methods,
including, for example, Western blotting using sucrose gradient purified-PRRSV
as antigen
(Mardassi etal., (1994) Can. J. Vet. Res. 58:55-64).
Blastogenic transformation test
In the blastogenic transformation test, cell proliferation is triggered by
antigen-exposure in
peripheral blood mononuclear cells (PBMC) isolated from pigs that have been
successfully
immunized with a PRRSV ORF.
At regular post-immunization intervals, pigs are medicated with Xylazine
(Bayers) at a dose of
lmg/Kg and blood samples collected from the anterior versa cava in vacuum
tubes containing
1/10 volume 150 mM sodium citrate in PBS, and then diluted I:3 in sterile
RPMI. Peripheral
blood mononuclear cells are separated by Ficoll-Paque (density 1.077;
Pharmacia)
centrifugation at 1,200 g for 20 min. The mononuclear cells are collected from
the huffy coat and
pelleted. The residual red blood cells are lysed by incubating cells with
0.53% ammonium
chloride for 10 min at 37C. After 2 washes in RPMI, the leukocytes are
adjusted to a suspension
of 2 X 106 cells per ml in RPMI containing 20% homologous heat inactivated
PRRSV negative
porcine serum, 50 U/ml of penicillin, and 50 pg/ml of streptomycin.
The antigen-specific (ORF-triggered) PBMC proliferation is determined by
incubating the
isolated PBMC in microtitration plates (4 X 10' cells in 200ft1/well in
triplicates) during 72 h in
the presence of variable concentrations ((l, 0.1, 10, and 25p,g/ml) of an ORF-
pH protein or an
effective fragment thereof. Blastogenic capacity of the PBMC under test
conditions is confirmed
by including positive control triplicates containing 2.5, 5, or 10 pg/ml of
the mitogen
31
CA 02418780 2003-02-28
Concanavaline A (ConA, Sigma Chemicals). After a 72 h stimulation period, the
cells are
labelled for 18 h with 0.1 pCi of [3H]thymidine (Amersham) per well, and
harvested with a
semiautomatic cell harvester (Skatron Instruments). The incorporated
radiolabelled nucleotide is
measured by scintillation counting after addition of a fluorescent liquid
scintillant (Cytoscint,
ICN). The level of proliferation is expressed as the mean of counts per minute
(CPM) of the test
wells minus the mean of the background CPM in control wells. Control for
background levels
consists of PBMC cultures in media alone.
Necropsy finding
Necropsy findings can also be compared in vaccinated and unvaccinated virus
challenged pigs
that have been euthanised. The respiratory tract, thoracic cavity and other
organs and tissues are
assessed by examining for both gross lesions and for microscopic lesions.
Histopathologieal examination
Thin sections (5 pm thick) of formalin-fixed, paraffin-embedded tissues from
the lungs, spleen,
liver, kidneys, and thoracic and mesenteric lymph nodes of pigs are routinely
processed for the
hematoxylin-phloxin-safran (HPS) staining, as described by Dea et al., (1991)
Journal of
Veterinary Diagnostic Investigation 3:275-282.
Virus isolation
In virus isolation, tissue samples are collected from various organs of pre-
vaccinated and/or
unvaccinated animals following viral challenge, and tissue homogenates are
inoculated onto host
cell monolayers, and these cells are then observed for cytopathic effects and
assayed for viral
protein by indirect immunofluorescence.
After collection of blood samples, pigs are euthanised by rapid intravenous
injection of sodium
pentobarbital (MTC Pharmaceuticals). Specimens are aseptically collected
various organs (for
example lungs, spleen, kidneys, liver, and mediastinal and mesenteric lymph
nodes). Tissue
homogenates are prepared in DMEM to final concentrations of 1:20 and 1:100.
Following
clarification by centrifugation at 10,000 g for 10 min, tissue homogenates are
inoculated onto
monolayers of cells, such as MARL-145 cells in 24 well-culture plates or PAMs
(porcine
alveolar macrophages) seeded in 96 well-microtitration plates. Cells are
harvested by 2 freeze-
thaw cycles at 4-5 days post-inoculation. Tissue culture supernatants are
clarified and used for a
second passage. Cultures are observed daily for cytopathic effects (CPE) until
day 5 post-
32
CA 02418780 2003-02-28
inoculation, at which time, infected monolayers are fixed with cold acetone
for indirect
immunofluorescence.
RT-PCR may also be used to reveal the presence of the viral genome in various
tissues from test
and control pigs. Total RNA is extracted from tissues collected from
challenged animals and
from cells, such as MARC-145 cells, inoculated with tissue homogenates. RT-PCR
is performed
using the oligonucleotide primers appropriate to amplify the desired ORF
region, as described by
Mardassi et al., (1995) Archives of Virology 140:1405-1418.
The occurrence of virus and/or viral CPE in a range of cultures (i.e.
inoculated with many
different tissue homogenates) is indicative of generalized viremia in the pig.
Homogenates from
successfully immunized animals might, for example, require a higher homogenate
concentration
for positive observation and/or show a delayed CPE (indicative of tow viral
titres), and/or
indicate a more localized viral presence, for example restricted mainly to the
respiratory tract.
Uses
The plasmid expression constructs of the present invention can be used as
transfer vectors to
construct other expression vectors, such as adenovirus expression constructs
described
previously.
Active Immunization
The compositions of the present invention can be used for the active
immunization of swine
against porcine reproductive and respiratory syndrome virus (PRRSV).
Immunization can be of
swine not yet exposed to the PRRSV virus, in which case the immunization can
confer prevent
the swine from becoming infected with the virus. It is also desirable to
immunize swine that
have or may have already been infected with the virus, to prevent or reduce
viral shedding.
Approaches to "sanitize" swine are known in the art.
The compositions of the present invention include vaccine compositions which
can be
administered in a conventional active immunization scheme: single or repeated
inoculations in a
manner compatible with the dosage formulation and in such amount as will be
prophylactically
effective and immunogenic, i. e. the amount of expression construct capable of
expressing a
codon-optimized ORF polypeptide that will induce immunity in an animal against
challenge by a
33
CA 02418780 2003-02-28
virulent PRRSV. Immunity is defined as the induction of a higher level of
protection in a
population of animals after vaccination compared to an unvaccinated group.
The amount of synORF polynucleotide, expression cassette or expression vector
to be introduced
into a vaccine recipient can be determined by a worker of skill in the art,
and will depend on the
strength of the transcriptional and translational promoters, and the type of
vector used, among
other things. In addition, the magnitude of the immune response will depend on
the level of
protein expression and the ability of the expressed ORF gene product to elicit
an immune
response.
Intramuscular injection, intraperitoneal or subcutaneous injection,
intradennal introduction,
impression through the skin, and other modes of administration such as
intraperitoneal,
intravenous, or inhalation delivery are also suitable. In an exemplary
embodiment,
administration is intramuscular or intradermal, for example injection at a
site behind the ear.
It is also contemplated that single or multiple booster vaccinations may be
provided.
The compositions of the present invention are advantageously administered in
the form of
injectable compositions either as liquid solutions or suspensions. Solid forms
suitable for
solution in, or suspension in, liquid prior to injection may also be prepared
if testing
demonstrates activity is retained in the solid form. A typical composition for
such purpose
comprises a pharmaceutically acceptable carrier. For instance, the composition
may contain 10
mg, 25 mg, 50 mg or up to about 100 mg of human serum albumin per millilitre
of phosphate
buffered saline. Other pharmaceutically acceptable carriers include aqueous
solutions, non-toxic
excipients including salts, preservatives, buffers, stabilizers (such as
skimmed milk or casein
hydrolysate), and the like. Examples of non-aqueous solvents are glycerol,
propylene glycol,
polyethylene glycol, vegetable oil, and injectable organic esters such as
ethyloleate. Aqueous
carriers include water, alcoholic/aqueous solutions, saline solutions,
parenteral vehicles such as
sodium chloride, Ringer's dextrose, etc. Intravenous vehicles include fluid
and nutrient
replenishers. Preservatives include antimicrobial agents, anti-oxidants,
chelating agents, and inert
gases. The pH and exact concentration of the various components of the
composition are
adjusted according to well-known parameters.
Adjuvant can also be included where an antigenic polypeptide is being
administered. Adjuvants
can conventional adjuvants as are known to workers skilled in the art, for
example
dimethyldioctadecyiammonium bromide in water (DDA) and sulfolipo-cyclodextrin
in squalene
in water. Adjuvants can alternatively or in addition be administered as
plasmids containing
immunostimulatory CpG motifs, and/or coding for immunostimulatory
polypeptides, for
34
CA 02418780 2003-02-28
example cytokines gamma interferon or interleukin-12 (see, for example, BS
McKenzie et al,
Immunol Res (2001) 24:225-44, Eo et al, Expert Opin Biol Ther (2001) 1:213-
25).
Passive Immunization
In accordance with the present invention, immunization can also be passive
immunization, in
which anti-PRRSV antibodies or serum containing such antibodies is
administered to an animal.
Using standard techniques known in the art, the plasmid expression constructs
of the present
invention can be used to inoculate swine, following which PRRSV-neutralizing
serum is
obtained from these swine. This serum can then be used for the passive
immunization of other
swine. Indeed, vaccinated pregnant sows could secrete large amounts of
specific anti-PRRSV
antibodies via their colostrum and milk which would protect suckling piglets.
Also, serum from
vaccinated animals, or purified gammaglobulin preparations, can be injected
parenterally
(intramuscular, intravenous or intraperitoneal injection) to naive or
immunodeprived pigs in
order to protect them temporarily from natural PRRSV infection.
Expressed proteins
The expressed recombinant proteins can also be used for the generation of
antibodies or as
antigens for the development of diagnostic procedures or using standard
techniques known in the
art.
Antibodies
For the production of antibodies and antibody fragments raised against a
target protein, various
hosts including goats, rabbits, rats, mice, humans, and others can be
immunized by injection with
the target protein, or with a fragment or oligopeptide thereof that has
immunogenic properties.
In accordance with the present invention, the target protein is a PRRSV
protein and the
oligopeptides, peptides, or fragments used to induce antibodies are encoded by
the codon-
optimized nucleic acid molecules and/or vectors of the invention.
Depending on the host species, various adjuvants may be used to increase
immunological
response. Such adjuvants include, but are not limited to, Freund's adjuvant,
mineral gels such as
aluminum hydroxide, and surface active substances such as lysolecithin,
pluronic polyols,
polyanions, peptides, oil emulsions, Keyhole limpet hemolysin (KLH), and
dinitrophenol.
Examples of adjuvants used in humans include, BCG (bacilli Calmette-Guerin)
and
Corynebacterium parvum.
CA 02418780 2003-02-28
The oligopeptides, peptides, or fragments used to induce antibodies can have
an amino acid
sequence consisting of as tittle as about 5 amino acids. In one embodiment of
the present
invention, amino acid sequences of at least about 10 amino acids are used.
These oligopeptides,
peptides, or fragments can be identical to a portion of the amino acid
sequence of the wild-type
protein that contains the entire amino acid sequence of a small, naturally
occurring molecule. If
required, short stretches of amino acids of the target protein can be fused
with those of another
protein, such as KLH, and antibodies to the chimeric molecule can be produced
Monoclonal antibodies to a target protein can be prepared using techniques
that provide for the
production of antibody molecules by continuous cell lines in culture. These
include, but are not
limited to, the hybridoma technique, the human B-cell hybridoma technique, and
the EBV-
hybridoma technique (see, for example, Kohler, G. et al. (1975) Nature 256:495-
497; Kozbor, D.
et al. (1985) J. Immunol. Methods 81:31-42; Cote, R. J. et al. (1983) Proc.
Natl. Acad. Sci. USA,
80:2026-2030; and Cole, S. P. et al. (1984) Mol. Cell Biol. 62:109-120). For
example, the
monoclonal antibodies according to the present invention can be obtained by
immunizing
animals, such as mice or rats, with purified protein. Spleen cells isolated
from the immunized
animals are then immortalized using standard techniques. Those isolated
immortalized cells
whose culture supernatant contains an antibody that causes an inhibition of
the activity of the
target protein with an ICS of less than 100 ng/ml are then selected and cloned
using techniques
that are familiar and known to one skilled in the art. The monoclonal
antibodies produced by
these clones are then isolated according to standard protocols.
The immortalization of the spleen cells of the immunized animals can be
carried out by fusing
these cells with a myeloma cell line, such as P3X63-Ag 8.653 (ATCC CRL 1580)
according to
the method in (1980) J. Imm. Meth. 39:285-308. Other methods known to a person
skilled in the
art can also be used to immortalize spleen cells. In order to detect
immortalized cells that
produce the desired antibody against the target protein, a sample of the
culture supernatant is
tested for reactivity using an enzyme linked immunosorbent assay (ELISA). In
order to obtain
those antibodies that inhibit the activity of the target protein, the culture
supernatant of clones
that produce antibodies that bind to the protein is additionally examined for
inhibition of protein
activity using an appropriate assay, such as those described herein. Those
clones whose culture
supernatant shows the desired inhibitory activity are expanded and the
antibodies produced by
these clones are isolated according to known methods.
1n addition, techniques developed for the production of "chimeric antibodies,"
such as the
splicing of mouse antibody genes to human antibody genes to obtain a molecule
with appropriate
antigen specificity and biological activity, can be used (Morrison, S. L. et
al. (1984) Proc. Natl.
36
CA 02418780 2003-02-28
Acad. Sci. 81:6851-6855; Neuberger, M. S. et al. (1984) Nature 312:604-608;
and Takeda, S. et
al. (1985) Nature 314:452-454). Alternatively, techniques described for the
production of single
chain antibodies can be adapted, using methods known in the art, to produce
single chain
antibodies specific to the target protein. Antibodies with related
specificity, but of distinct
idiotypic composition, can be generated by chain shuffling from random
combinatorial
immunoglobulin libraries (see, for example, Burton D. R. (1991) Proc. Natl.
Acad. Sci. USA,
88:10134-10137).
Antibodies can also be produced by inducing in vivo production in the
lymphocyte population or
by screening immunoglobulin libraries or panels of highly specific binding
reagents as disclosed
in the literature (Orlandi, R. et al. (1989) Proc. Natl. Acad. Sci. 86: 3833-
3837; Winter, G. et al.
( 1991 ) Nature 349:293-299).
Antibody fragments which contain specific binding sites for the target protein
can also be
generated. For example, such fragments include, but are not limited to,
F(ab')2 fragments
produced by pepsin digestion of the antibody molecule and Fab fragments
generated by reducing
the disulphide bridges of the F(ab')2 fragments. Alternatively, Fab expression
libraries can be
constructed to allow rapid and easy identification of monoclonal Fab fragments
with the desired
specificity (see, for example, Huse, W. D. et al. (1989) Sciene,e 246:1275-
1281).
Various immunoassays can be used for screening to identify antibodies having
the desired
specificity. Numerous protocols for competitive binding or immunoradiometric
assays using
either polyclonal or monoclonal antibodies with established specificities are
well known in the
art. Such immunoassays typically involve the measurement of complex formation
between the
target protein and its specific antibody. Examples of such techniques include
ELISAs,
radioimmunoassays (RIAs), and fluorescence activated cell sorting (FRCS).
Alternatively, a two-
site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to
two non-
interfering epitopes, or a competitive binding assay can be used (see, Maddox,
D. E. et al. (1983)
J. Exp. Med. 158:1211-1216). These and other assays are well known in the art
(see, for
example, Hampton, R. et al. (1990) Serological Methods: A Laboratory Manual,
APS Press, St
Paul, Minn., Section IV; Coligan, J. E. et al. (1997, and periodic
supplements) Current Protocols
in Immunology, Wiley & Sons, New York, N.Y.; Maddox, D. E. et al. (1983) J.
Exp. Med.
158:1211-1216).
Diagnostics
The expression constructs of the present invention can also be used for
diagnostic purposes.
Recombinant adenoviruses or recombinant plasmid DNA vectors carrying the codon-
optimized
37
CA 02418780 2003-02-28
PRRSV ORF polynucleotide may be used to infect or transfect cell cultures
(e.g. COS7, 293,
A549, MARL-145, or KB cells) resulting in high levels of expression of the ORF
protein
carrying major antigenic determinants in the cell cultures. These cells
cultures can be used for
diagnostic purposes; for example, one can determine whether an animal contains
specific
S antibodies in its serum that are directed to the expressed ORF protein using
immunochemical
techniques such as indirect immunofluorescence, immunoperoxidase, immunogold
silver
staining, or enzyme linked immunosorbant assay (ELISA). The recombinant ORF
protein can
also be recovered from the supernatant fluids of homogenates of the adenovirus-
infected cell
cultures and used as a source of antigens for ELISA, radioimmunoassay (RIA),
and agglutination
assays.
Standard nucleic acid techniques known in the art can be used in combination
with at least one
primer specific to a synORF polynucleotide sequence of the invention, to
detect the presence of
that synORF polynucleotide in a pig previously imnwnized with that synORF. In
general, a
biological sample, for example a tissue or a bodily fluid, is first obtained
from the pig. The cells
within the sample can be lysed and the crude lysate used in the assay or
nucleic acids can be
isolated from the sample. The nucleic acids can be used directly in the assay
or they can be
subjected to an initial amplification step. The present invention also
contemplates the adaptation
of the diagnostic screening to high-throughput technology.
A nucleic acid detection procedure can be used to verify expression of the
synORF sequence
used in immunization. It is also in accordance with the present invention to
use the results of
such an assay in conjunction with an assay to detect the presence of nucleic
acid sequences
representing PRRSV infection in an immunized animal. When both tests are run
on the same
sample (potentially, but not necessarily on different aliquots of the same
sample) primer
sequences can be chosen so that the amplified sequences will be specific to
each of the synORF
polynucleotide and the infectious PRRSV agent. Alternatively primers used in
an assay to detect
potential PRRSV infection in an animal previously immunized with a synORF
polynucleotide
can be targeted to a sequence not present in the synORF polynucleotide so as
to avoid false
positive results. That is, at least one primer can be chosen that does not
hybridize specifically
with the synORF polynucleotide used to immunize the pig. The assay conditions
under which
the ability of a primer and polynucleotide to specifically hybridize may be
tested are well known
in the art, and require relatively stringent hybridization conditions.
38
CA 02418780 2003-02-28
Primers
Primers for use in synthesis or in assays to detect in a sample the presence
of codon-optimized
nucleic acid molecules of the invention can be designed and made using
standard techniques
known by a worker skilled in the art. To generate a specific synthetic or
diagnostic result, at least
one of each pair of primers used in an amplification technique such as PCR is
specific to the
target polynucleotide. As is understood by a worker skilled in the art,
primers are designed and
selected based on their ability to hybridize with a level of specificity
appropriate to the
application. Nucleic acid hybridization will be affected by the base
composition, length of the
complementary strands, and the number of nucleotide base mismatches between
the hybridizing
nucleic acids, in addition to such conditions as salt concentration,
temperature, and solvents, as
will be readily appreciated by those skitled in the art.
Primers for use in the synthesis of codon-optimized nucleic acid molecules of
the invention are
shown in Table 5 and contain sequences specific to the synORFS polynucleotide.
Primers for
use in diagnostic assays include these primers, or fragments of these primers
that are from 15-30,
or 18-25, or 20-23 nucleotides long. A diagnostic assay that distinguishes
between the presence
of a sequence encoding a PRRSV ORF (e.e. present due to a natural PRRSV
infection) and a
codon-optimized ORF (e.g. present in a sample due to inoculation with a synORF
nucleic acid of
the invention), will employ primers that detect the synORF or fragments
thereof but not t the
wtORF nucleotide sequence. Examples of such primers, for the detection of
synORFS, include
(nucleotide numbers refer to the synORFS sequence of Figure 2 and SEQ ID NO:
1):
Primers (sense or antisense) comprising at least a 14-mer sequence
corresponding to nt
94-107 at the most 3' end, e.g. a 18-mer sense primer having the sequence of
nt 90-107,
or a 20-mer antisense primer capable of hybridizing to nt 111-94.
A 20-mer sense primer containing at least a sequence corresponding to nt 310-
329, i.e.
having the sequence 5'- GGCCGCTACGTGCTGTCCTC -3'.
Primers (sense or antisense) comprising at least a 16-mer sequence
corresponding to nt
573-588 at the most 3' end, e.g. a 20-mer sense primer having the sequence of
nt 569-
588, or a 20-mer antisense primer capable of hybridizing to nt 573-592.
Primers (sense or antisense) comprising at least a 16-mer sequence
corresponding to nt
372-387 at the most 3' end, e.g. a 20-mer sense primer having the sequence of
nt 368-387
or a 20-mer antisense primer capable of hybridizing to nt 372-391.
39
CA 02418780 2003-02-28
Primers (sense or antisense) comprising at least a 14-mer sequence
corresponding to nt
36-49 at the most 3' end, e.g. a 18-mer sense primer having the sequence of nt
32-49 or a
18-mer antisense primer capable of hybridizing to nt 36-53.
Thus, for example, a synORFS primer set could include the sense
oligonucleotide primer for the
sequence of nucleotides 310-329, in combination with the antisense
oligonucleotide primer for
the sequence of nucleotides 592-573, yielding an amplified fragment 283
nucleotides long. A
second exemplary synORFS primer set could include the sense primer having the
sequence of
nucleotides 90-107 in combination with the antisense primer capable of
hybridizing to synORFS
nucleotides 331-312, which would generate a PCR fragment 242 nucleotides long.
Kits
A kit can be assembled comprising some or all of the essential materials and
reagents required
for vaccinating swine with polynucleotides of the invention or antiserum
raised against
polynucleotides of the invention, for constructing expression vectors encoding
synORF
polynucleotides, for transforming cells with expression vectors of the
invention, for detecting
synORF polynucleotides of the invention, or for detecting PRRSV infection.
This generally will
comprise selected expression constructs, andlor anti-PRRSV ORF antiserum or
specific or
monoclonal antibodies, and/or primers. Also included may be various media for
replication of
the expression constructs and host cells for such replication. Such kits will
typically comprise
distinct containers for individual reagents.
When the components of the kit are provided in one or more liquid solutions,
the liquid solution
can be an aqueous solution, for example a sterile aqueous solution. For in
vivo use, the
expression construct may be formulated into a pharmaceutically acceptable
syringeable
composition. In this case the container means may itself be an inhalant,
syringe, pipette, eye
dropper, or other such like apparatus, from which the formulation may be
applied to an infected
area of the animal, such as the lungs, injected into an animal, or even
applied to and mixed with
the other components of the kit.
The components of the kit may also be provided in dried or lyophilized forms.
When reagents or
components are provided as a dried form, reconstitution generally is by the
addition of a suitable
solvent. It is envisioned that the solvent also may be provided in another
container means.
Irrespective of the number or type of containers, the kits of the invention
also may comprise, or
be packaged with, an instrument for assisting with the
injection/administration or placement of
the ultimate complex composition within the body of an animal. Such an
instrument may be an
CA 02418780 2003-02-28
inhalant, syringe, pipette, forceps, measured spoon, eye dropper or any such
medically approved
delivery vehicle suited to use in the instant invention.
To gain a better understanding of the invention described herein, the
following examples are set
forth. It should be understood that these examples are for illustrative
purposes only. Therefore,
they should not limit the scope of this invention in any way.
41
CA 02418780 2003-02-28
EXAMPLES
FXAMPI.F 1
Materials and methods
PRRS Virus and cells
The IAF-Klop strain of PRRSV has been found to be highly pathogenic for MARL-
145 cells, a
clone of MA-104 cells highly permissive to PRRSV (Kim et al., 1993), as
previously described
(Mardassi et al., 1994). PRRSV can also be propagated effectively on primary
cultures of
pulmonary alveolar macrophages, as well as in blood monocytes (Therrien et
al., (2000) Arch
Virol. 145:1099-116).
Isolation of propogated PRRSV
To eliminate cellular membranes and debris from viral suspension, lysates of
the infected cells
(approximately 200 ml) are clarified by centrifugation at 8,000 X g for 15
min. Then, the viral
particles in the supernatant fluid are concentrated by differential
ultracentrifugation through a
cushion of 30% sucrose (wt/wt) in 0,05M Tris-buffered saline, pH 7.0 to 7.5
(TBS). Thereafter,
the viral pellet is gently dispersed, diluted in 1 or 2 ml of TBS for an
isopycnik
ultracentrifugation through a 1.2 tol.5 CsCI continuous density gradient. The
opalescent viral
bands are recovered in the fractions corresponding to a density of 1.18 to
1.20 g/ml of CsCI
(Mardassi et al. (1994) Can. J. Vet. Res. 58:55-64). Negative stain electron
microscopy can be
used to reveal the presence and morphology of viral particles.
Adenovirus vectors and cells
Ad/CMVIacZ (Acsadi et al., 1994), a replication-defective E1- and E3-deleted
hAdVS, as well as
generated hAdVs, were propagated in 293 cells (ATCC CRL-1573), to complement
the functions
of the E1-deleted genomic region of Ad/CMVIacZ and thereby permit the
replication of
replication-defective hAdVs. Infectivity titres of hAdVs were determined by
calculation of the
plaque forming units (PFU/ml) on 293 cell monolayers, as detailed elsewhere
(Massie et al. ,
1998c). AdCMV/tTA permits the constitutive expression of the tetracycline
transactivator (tTA)
in infected cells using the constitutive human CMV immediate-early
promoter/enhancer. The
tTA is essential to allow expression in hAdV-infected cells of the foreign
gene that has been
cloned downstream and under the control of the TR5 promoter. Doxycycline
(Sigma), an
42
CA 02418780 2003-02-28
analogue of tetracycline was used at a concentration of 1 pg/ml to inhibit the
expression of the
foreign gene in hAdV-infected cells (Massie et aL, 1998b). The 293 TetOn cells
(Clonetech Inc)
are 293 transformed cells that constitutively express the reverse tetracycline
transactivator
(rtTA). These cells were cultivated in the presence of 1 pg/ml of doxycycline
to enhance the
expression by hAdVs of the transgene placed under the control of the
tetracycline-regulable
promoter (TR5) (Massie et al., 1998b). BMAdEl cells, an A549 cell line
expressing AdEI
proteins from a vector designed to eliminate the generation of replication
competent hAdVs
(Massie, 1998x), were propagated in the same conditions as the 293 cells
(Acsadi etal., 1994).
Design and construction of the synthetic ORFSgene of PRRSV
Tables 3a-c show the colon usage of the ORFS, ORF4 and ORF6 proteins of the
IAF-Klop strain
of PRRSV, in comparison with colon usage in highly expressed human (H) genes.
The
frequencies (x 100) of the individual colons are shown for each of the
degenerately encoded
amino acids, as well as the number of each amino acids for the PRRSV protein
in parenthesis,
and the most prevalent colon is shown in bold. The sequence of a synthetic
ORF5
polynucleotide encoding a wild-type amino acid sequence (SEQ ID NO: 2) is
shown in Figure 2
and SEQ ID NO: 1. The colons most frequently used by mammalian cells were used
in the
synORFS gene according to Haas et al. (1998). In the construction of the
completely optimized
synthetic ORFS polynucleotide (synORF.S) of Figure 2 (SEQ ID NO: 1), a total
of 130
nucleotides were optimized over the entire IAF-Klop ORFS sequence.
To construct the synORFS, 21 long synthetic oligonucleotides of 20-60 mers
covering the entire
ORFS gene of the IAF-Klop strain of PRRSV with 30 mer overlaps were
synthesized using an
automated synthesizer (Pharmacia Biotech Inc., Baie d'Urfe, Quebec) (Table 5).
The synORF5
gene was assembled by single overlap PCR, as described by Holler et al.,
((1993) Gene 136:323-
328). The PCR reactions were performed in 50-~l reaction mixture containing
each
deoxynucleoside triphosphate at a concentration of 0.2 mM, plus 50 pmol of
each forward and
reverse primer, 20mM Tris-HCI, 50 mM HCI, 1.5 mm MgCl2 (GIBCO BRL), and 10 U
of Taq
DNA polymerase (GIBCO BRL). The PCR amplifications were performed in a DNA
Engine
Thermocycler (MJ Research model PTC-100, with hot bonnet) using the following
protocol: 33
amplification cycles of denaturation at 94 °C for 60 s, primer
annealing at 55 °C for 60 s, and
elongation at 72°C for 90 s, followed by a final extension step at 72
°C for 10 min. Aliquots of
10 p1 of the amplified products were visualized by electrophoresis on 1,5%
agarose gels in TAE
buffer (0.04 M Tris-acetate pH 8.5, 0,002 M EDTA ) in the presence of ethidium
bromide at 100
V for 1 h and then visualized under UV illumination. The bands of the expected
size were cut
43
CA 02418780 2003-02-28
and the DNA was purified by using the QiaGen DNA Extraction kit (QiaGen Inc.,
Mississauga,
ON, Canada). The purified DNA was then used in the next step of extension of
the gene. The
final PCR product containing the entire synORFS gene with A overhangs was
cloned into
pCR2.1 vector using Topo-TA cloning kit (Invitrogen Co., San Diego, CA),
according to the
S manufacturer's directions.
In an alternative approach, the entire wtORFS and synORFS genes were cloned
into pRc/CMV2
(Invitrogen) eukaryotic vector by adding Hind III and Xba I restriction sites
at the 5' ends of the
sense and antisense oligonucleotide primers, respectively. The resultant
recombinant plasmids
were digested with BamHl or Hind III endonucleases to verify the size of
cloned DNA fragment.
The nucleotide sequence of the wtORFS and synORFS genes were verified by
sequencing both
strands by the dideoxynucleotide chain-termination method (Sanger et al.,
1977) using the T7
DNA polymerase (Pharmacia) in an Automated Laser Fluorescent DNA sequencer
(Pharmacia
LKB). To assess the error rate of the reverse transcriptase and Tag
polymerase, clones from three
different PCR events were sequenced. Subsequently, the nucleotide (nt) and
amino acid
sequences were computer analyzed with the GeneWorks 2.4 program
(IntelliGenetics Inc.,
Mountain View, Calif.). Corrections of the errors made in the synORFS were
made by other runs
of PCR using as template the synORFS cloned into the pRc/CMV2 plasmid, using
the DNA
Vents polymerase (New England Biolabs) and sense and antisense primers
corresponding to the
regions where the errors occurred.
Antisera
Rabbit monospecific a5-hyperimmune serum to E. coli-expressed ORFS product of
the
homologous PRRSV strain was obtained from previous studies (Mardassi et al.,
1996). The
hyperimmune porcine anti-PRRSV serum was obtained following experimental
inoculation of
SPF piglets (Loemba et al., 1996).
Cloning of the nazi ve ORFS and synORFS genes in eukaryotic plasmids for
transient expression
experiments.
Viral RNA was extracted from PRRSV-infected MARC-145 cells by the one-step
guanidinium
isothiocyanate-acid phenol method (Chornczynski & Sacchi, 1987). The native
ORFS encoding
region, as well as synORFS within the pCR2.1 vector, were cloned into the Hind
III and Xba I
cloning sites of the eukaryotic expression vector pRc/CMV2 (Invitrogen),
downstream of the
human cytomegalovirus (HCMV) promoter to produce pRc/CMV2/wtORF.S and
44
CA 02418780 2003-02-28
pRc/CMV2/synORFS recombinant piasmids. The sequences of the oligonucleotide
primers used
for the latter amplification were as follows:
ETS 5': 5'- CC GGATCC GCC GCC GCC ATG TTG GGG AAA TGC CTG ACC- 3', (SEQ ID
NO: 5) or
ETS 5' syn: 5'- CAT GGATCC GCC GCC GCC ATG CTG GGC AAG TGC TTG ACC- 3'
(SEQ ID NO: 6)
which are forward primers that comprise the first ATG codon of the wtORFS (ETS
S') and
synORFS (ETS 5' syn) genes downstream of a Kozak motif for initiation of
translation in
vertebrates (Kozak, 1987), and
ETRS: 5'- TCTAGA GGCAAAAGTCATCTAGGG-3' (SEQ ID NO: 7)
a reverse primer which comprises the C-terminal stop codon of the viral gene.
The nucleotide
sequence accession number (EMBL/GenBank/DDBJ libraries) of ORFS of the IAF-
Klop strain is
U64928 (Gagnon & Dea, 1998). For directional cloning, Hind III and Xba I
restriction sites were
added at the 5' ends of the sense and antisense oligonucleotide primers,
respectively, and the
synORFS gene cloned into the corresponding Hind III and Xba I sites of the
expression vector
pRc/CMV (Invitrogen) downstream of the human cytomegalovirus (CMV) promoter,
producing
the plasmid pRc/CMV2/synORFS. Both strands of pRc/CMV2/wtORFS and
pRc/CMV2/synORFS were sequenced in an Automated Laser Fluorescent DNA
sequences
(Pharmacia LKB) in order to confirm that no error has occurred as a result of
PCR amplification.
Transient expression of the GPSglycoprotein
Ex-vivo expression of pRc/CMV2/wtORFS and pRc/CMV2/synORF5 constructs were
tested in
transient expression experiments in cells maintained as confluent monolayers.
Cells in 6 cm-
tissue culture plates were transfected with 15 pg of plasmid DNA using Fugene
6 transfection
reagentTM (Roche Diagnostics, Laval, Qc, Canada) and incubated at 37
°C. For indirect
immunofluorescence (IIF), cells were rinsed twice in PBS and fixed with 80%
cold acetone for
20 min at 4 °C at variable times (18 to 72 h) post-transfection. The
monolayers were then reacted
for 30 min with rabbit monospecific a5-hyperimmune serum (Mardassi et al.,
1996) and the
immune reaction was revealed following incubation with fluorescein-conjugated
goat anti-rabbit
Ig (Roche Diagnosis Ins., Laval, Canada), as previously described (Loemba et
al., 1996).
CA 02418780 2003-02-28
Generation of recombinant replication-defective hAdVs expressing the PRRSV
native ORFS and
synORFS genes.
The entire wtORFS (SEQ ID NOs: 3 and 4) and synORFS genes (SEQ ID NOs: 1 and
2, or 8 and
9) of the IAF-Klop strain of PRRSV were amplified by RT-PCR using specific
sets of
oligonucleotide primers which have been designed from the previously described
sequence of the
virus (EMBL/Genbank accession No. U64928: Gagnon & Dea, 1998; Pirzadeh et al.,
1998).
Both primers contained two BamHI restriction sites at their 5' end, and in the
case of sense
primer, the ATG initiator codon was preceded by a triple GCC motif in order to
provide an
optimal Kozak consensus sequence for efficient translation (Kozak, 1987). For
each reaction, the
amplified product was inserted into the unique BamHI site of the adenovirus
transfer vectors
pAdTRS/DCIGFPq (Massie et al., 1998c) so that the wtORFS and synORFS coding
sequences
would be under the control of the TR.S promoter (Massie et al., 1998b).
The recombinant plasmids were linearized by digestion at the unique Cla I site
and rescued into
the genome of Ad/CMVIacZ, a replication-defective E1- and E3-deleted hAdVs, by
homologous
recombination in 293 cells, as described elsewhere (Jani etal., 1997). The 293
cells, were used to
propagate hAdVs to complement the functions of the E1-deleted genomic region
of
Ad/CMVIacZ and thereby permit the replication of replication-defective hAdVs.
Upon
cotransfection, virus plaques were isolated, amplified in 293 cells, and
analyzed for the
expression of the recombinant GPS protein either by western blotting or by
radioimmunoprecipitation assays (RIPA). The hAdVs AdTRS/DC/GFPq/wtORFS
(hAdV/TRS/wtORFS), and AdTRS/DC/GFPq/synORFS (hAdV/TR5/synORFS), which
efficiently evoked the expression of the GPS , were subjected to three
consecutive rounds of
plaque purification on BMAdEl clone 78, then selected viral clones were
amplified on BMAdEl
clone 220 cells (up to 3X10' cells), as previously described (Massie et al.,
1998a). Infectivity
titres of the hAdVs were determined by calculation of the plaque forming units
(PFU/ml) on 293
cell monolayers, as detailed in Massie et al. (1998b).
Western blotting experiments
Lysates of MARC-145 cells, infected with PRRSV, or infected with
hAdV/TRS/wtORFS or
hAdV/TRS/synORFS together with hAdV/CMV/tTA, or with hAdV/CMV/tTA alone, were
prepared in LB-2 lysis buffer (Mardassi et al. , 1996) and denatured by
boiling in the presence of
S% (V/V) ~3-mercaptoethanol, subjected to 12% SDS-PAGE and electrotransferred
onto
nitrocellulose membranes (45 pm pore size, Schleicher and Schuell Inc.)
(Loemba et al., 1996).
Immunological identification of native or recombinant viral proteins was
confirmed following
46
CA 02418780 2003-02-28
incubation of the saturated nitrocellulose membranes in the presence of 1:100
to 1:1000 dilution
of the rabbit monospecific a5-hyperimmune serum or hyperimmune porcine anti-
PRRSV serum,
as previously described (Mardassi et al. , 1994; 1996).
Metabolic labeling and immunoprecipitation of PRRSV native or recombinant
proteins
Radiolabelling with (35S]-methionine (specific activity of 1,120 Ci/mmole,
Amersham Searle
Co., Oakville, Ontario) of viral proteins synthesized in PRRSV-infected MARC-
145 cells, as
well as recombinant proteins synthesized in 293 or 293 TetOn cells infected
with hAdVs, was
carried out essentially as previously described (Mardassi et al., 1994, 1996).
These cells were
cultivated in the presence of 1 ~g/ml of doxycycline (Sigma chemical Inc., St-
Louis, Mo) to
enhance the expression by hAdVs of the foreign genes placed under the control
of the TRS
promoter (Massie et al., 1998c). Aliquots (adjusted to 1 x 10'cpm per 500 p1
of RIPA buffer) of
clarified lysates of PRRSV-infected, hAdVs-infected or mock-infected cells
were incubated
overnight at 4°C with 5 to 15 p1 of the rabbit aS monospecific
antiserum or anti-PRRSV
hyperimmune pig serum. The immune complexes were then adsorbed for 2 h to
protein A-
sepharose CL4B beads (Amersham Inc.) and dissolved directly in electrophoresis
sample buffer
containing 5% (3-mercaptoethanol. Following electrophoresis on 12,5 % SDS-
polyacrylamide
gels, the immune complexes were revealed by fluorography and autoradiography,
as previously
described (Dea et al., 1989).
Animals
Nine crossbred F1 (Landrace x Yorkshire) castrated specific pathogen-free
(SPF) piglets 4 to-5
week of age were obtained from a breeding farm located in southern Quebec,
Canada. The
breeding stock and piglets were tested and proven to be seronegative for
PRRSV,
encephalomyocarditis virus (EMCV), porcine parvovirus (PPV), haemagglutinating
encephalo-
myelitis virus (HEV), transmissible gastroenteritis virus (TGEV) and
Mycoplasma
hyopneumoniae. The piglets used in this study were from 2 different litters
and were randomly
divided into one control group and two experimental groups (3 piglets /group)
kept in facilities
equipped with a microorganism-free, filtered in-flowing and out-flowing air
system. The animals
were fed with commercial feed and water ad libitum.
Pig immunization protocol and challenge
47
CA 02418780 2003-02-28
Groups of 3 piglets were given two injections, 32 days apart, of 1) a volume
of 100 p.1 of a
suspension containing 109PFU of hAdV/TR5/wtORFS mixed with 5 x 109 PFU of the
hAdV/CMV/tTA in PBS containing 0,02% of the poloxamer SP1017 (Lemieux et al.,
2000)
(Suprateck Pharma Inc., Laval, QC, Canada); 2) a volume of 100p,1 of a
suspension containing 1
x lO~PFU of hAdV/TR5/synORFS mixed 1:5 with hAdV/CMV/tTA prepared in the
mixture
described above; or 3) a volume of 100 pL of a suspension containing 5 x 10 9
PFU of
hAdV/CMV/tTA prepared in the SP1017 poloxamer solution (control pigs). The
suspensions of
hAdVs were injected intradermally under the right ear using a 30 gauge needle.
The animals
received a booster of the same antigenic mixture at day 32, and were
challenged intranasally at
day 60 with a dose of 105 TCIDS~ of the IAF-K(op strain in 5 ml of clarified
cell culture
supernatant. Pigs were bled at days 0, 10 and 21 post-challenge.
Virus neutralization and serological tests
Pig sera were tested for the presence of specific anti-GP; antibodies by virus
neutralization (VN),
IIF, ELISA and Western blotting (WB) tests. The VN test was performed in
triplicates with 100
p1 of serial dilutions of heat-inactivated (56 °C, 45 min) test sera,
incubated for 60 min at 37 °C in
the presence of 100 TCIDSO of the virus in DMEM containing 20% normal SPF pig
serum (Yoon
et al. , 1994). The mixtures were put in contact with confluent monolayers of
MARL-145 cells in
96 well microtitration plates, incubated at 37 °C in a humidified
atmosphere containing 5% COZ,
and observed daily for up to 5 days for the appearance of cytopathic effects
(CPE) (Loemba et
al., 1996). The monolayers were then fixed with a solution of 80% cold acetone
in PBS buffer,
and tested for expression of the PRRSV nucleocapsid protein by IIF (Magar et
al. , 1995), using
N protein specific MAb IAF-K8 (Dea et al., 1996). The immune reaction was
visualized after an
incubation of 45 min. with FITC-labelled goat anti-mouse IgG (Roche Diagnosis
Inc.).
Neutralizing titres were expressed as the reciprocal of the highest dilution
that completely
inhibited the expression of viral N protein. A competitive ELISA for detection
of antibodies to
PRRSV using recombinant E. coli-expressed N protein as antigen was used to
monitor humoral
immune response following challenging of hyper-immunized pigs (Dea etal.,
2000b). A
commercial indirect ELISA (Idexx) for detection of anti-PRRSV antibodies was
also used,
following the manufacturer's directions. Western blotting was performed as
described above,
using sucrose gradient purified-PRRSV as antigen (Mardassi et al., 1994).
Results
48
CA 02418780 2003-02-28
Construction of a synthetic ORFS gene based on optimal codon usage
Initially, PCR was adopted for multiple simultaneous single-overlap extension
for gene assembly
by mixing a series of internal oligonucleotides designed to alternate in
sequence on the sense and
antisense strands, together with an excess of flanking primers in one reaction
mix. No product of
the expected size of 603-by could be detected by agarose gel electrophoresis
of the initial
reaction mixture. Changes in oligonucleotide concentration and thermal cycling
parameters did
not improve upon this result. In an alternative approach, the most C-terminal
I80-by was first
amplified, and the gene was then extended by next PCR amplification mixing the
upstream
overlap oligonucleotides and the previous PCR product. Two short
oligonucleotides flanking the
entire ORFS gene were used to amplify the whole synthetic gene (Gonin et al.,
1999).
The first clone obtained was totally sequenced. A total of 14 errors were
detected in the 603
nucleotide sequence to give an overall error rate of one per 58 nucleotides.
This high error rate
was probably due to the numerous cycles of amplification using Tag polymerase.
Eleven of the
errors corresponded to nucleotide substitutions occurring at wobble base, thus
leading to silent
amino acid mutations (amino acid residues unchanged). Three errors resulted
from single by
substitutions leading to amino acid changes: G for A at position 143, and G
for T at both
positions 187 and 464. These by substitutions correspond to amino acid changes
at positions 47
(Cys for Tyr), 62 (Ala for Ser) and 155 (Try for Leu) of the authentic GPS
envelope glycoprotein,
respectively. Four silent mutations occurred within the first 204 by of the
entire cDNA and were
corrected by repeating PCR of that region. This corrected portion of the cDNA,
with the
exception of the A and T at positions 143 and 155 was then assembled with the
remaining
portion of cDNA, with the single non-silent error at position 464, to obtain
the preliminary entire
synORFS gene. After a final PCR amplification using ET5'syn and ETRS primers
pair, the
temporary uncorrected synORFS was inserted into the eukaryotic pRc/CMV2
expression plasmid
and used as DNA template for final correction by PCR using the appropriate 60-
mer
oligonucleotides as primers and the Vent' DNA polymerase (New England Biolab).
The parental recombinant plasmid (pRcICMV2IsynORFS) was used in its circular
form. In a
first step, an appropriate primer pair was used to obtain a first PCR product
corresponding to
DNA of the entire parental plasmid containing the synORFS with targeted
mutations G143A and
GI87T. The advantage in obtaining an amplified product with blunt ends permits
recirculation of
the plasmid by simple ligation, the latter being used to transform E. coli
competent cells (DHSa
strain) for amplification and verification of the corrections by sequencing
analysis. This latter
recombinant plasmid was in turn used for reverse PCR using a second primer
pair to correct the
mutation G464T. Final sequencing analysis of the inserted synORFS indicated
that the errors
49
CA 02418780 2003-02-28
have been corrected (Figure 2). The final construct of synORFS contains a
total of 130
nucleotide substitutions compared to the wtORFS gene, resulting in an overall
78,4 % (473/603)
identity at the nucleotide Level, but deduced amino acid sequences from both
wtORFS and
synORFS genes were 100 % identical. Thus, a new gene coding for the major GP5
envelope
glycoprotein of a North American strain of PRRSV, the IAF-Klop strain, was
successfully
created.
Transient expression of wild type and synthetic ORFSgenes in 293 and MARC-145
cells
Ex-vivo expression of pRc/CMV2/wtORFS and pRe/CMV2/synORFS constructs were
tested in
transient expression experiments in cells maintained as confluent monolayers.
The synthesis of
GPS in both human 293 and simian MARL-145 cells was confirmed by indirect
immunofluorescence (IIF) following incubation of cells transfected with the
recombinant
eukaryotic plasmids in the presence of monospecific a5- rabbit hyperimmune
serum and the
appropriate fluorescein-labelled goat anti-rabbit Ig conjugate. In both cases,
the optimal number
of transfected cells, as well as the optimal intensity of the fluorescence,
were observed at 48 h
post-transfection (Figure 3). A specific cytoplasmic fluorescence of weak
intensity was observed
in approximately 5 to 10% of the cells transfected with the wild type gene,
while up to 20 to 25%
of the synORFS-transfected cells displayed a more intense cytoplasmic
fluorescence that tended
to accumulate near the perinuclear region. Surprisingly, both the number of
cells transfected
with the synORFS recombinant plasmid, and the intensity of fluorescence per
cell, were higher.
Generation of inducible replication-defective adenoviral vectors for the
expression of synORFS
gene
In MARC-145 cells, as well as in 293 cells, co-infection of hAdVs
constitutively expressing the
tTA transactivator (AdCMV/tTA), and hAdVs expressing the PRRSV major GP5
envelope-
associated gene under the control of the tetracycline-regulated promoter
(hAdV/TR5/wtORFS or
hAdV/TR5/synORFS), allowed efficient controlled expression of the PRRSV
recombinant
protein. The addition of doxycycline in the medium completely abrogated
expression of the
transgenes (Gagnon et al., 2001, 2003), confirming that the tetracycline
regulated expression
system was effective.
To facilitate the identification of the recombinant clones or hAdVs, the gene
encoding for the
GFPq protein (Green fluorescent protein) was cloned as a reporter gene
downstream of the
multiple cloning site of the shuttle vector under the control of an internal
ribosomal entry site
(IRES). As illustrated in Figure 4, at 24, 48 and 70 h post-infection at a
multiplicity of infection
CA 02418780 2003-02-28
(moi) of 100 PFU per cell, the intensity of the spontaneous GFPq fluorescence
was higher in
cells infected with the recombinant hAdVs expressing the synORFS gene than
those expressing
the wtORFS gene. This suggests that, suprisingly, transcription of the synORFS
gene was more
efficient than that of the wtORFS gene, thus allowing a higher interaction of
the GFPq gene with
cellular enzymes involved in mRNAs synthesis and ribosomes for translation.
In agreement with these findings, expression of the GP5 major envelope
glycoprotein per se was
also higher in cells infected with synORFS than those infected with wtORFS.
MARC-145 cell
monolayers were co-infected with AdCMV/tTA (control lane) or AdCMV/tTA and
hAdV/TRS/DC/GFPq/wtORFS (lane WT) or hAdV/TR5/DC/GFPq/synORFS (lane SYNT) at a
moi of 100 PFU. After incubation for 24 or 48 h (Figure 5) or 70 h (not shown)
cells were fixed
with cold acetone and washed twice with PBS to eliminate spontaneous GFPq
fluorescence.
Expression of GPS of PRRSV was confirmed by specific IIF following incubation
in the presence
of the rabbit anti-a5 monospecific serum. The intensity of the cytoplasmic
fluorescence was
optimal at 48 h post-infection (Figure 5).
When cultivated in 293 TetOn cells (cells that constitutively express the
reverse tetracycline
transactivator (rtTA) thus repressing expression in the absence of
doxycycline) the presence of 1
pg/ml of doxycycline enhanced the expression of GP5 from both hAdV/TRS/wtORFS
and
hAdV/TRS/synORFS vectors (data not shown).
Expression of the wild type and synthetic GPS as revealed by radio-
immunoprecipitation
In order to correlate data obtained by immunofluorescence with the levels of
synthesis of the GP5
major envelope glycoprotein of PRRSV, RIPA experiments were conducted with
lysates of 293
rtTA cells infected with either hAdV/TR.S/wtORFS or hAdV/TR5/synORFS
recombinant
viruses.
The immune complexes obtained after incubation in the presence of rabbit
monospecific a5-
hyperimmune serum were adsorbed on protein A-sepharose beads, then analysed by
SDS-PAGE
and revealed by fluorometry and autoradiography. As shown in Fig 6 (left and
right panels),
immunoprecipitation of cell lysates harvested 48 h post-infection, revealed an
increased
expression of GP5 in cell cultures infected with hAdV/TR5/synORFS in
comparison to the level
of GP5 synthesized in cell cultures infected with hAdV/TR5/wtORFS. Using
densitometry it was
determined that the amount of the PRRSV GP5 major envelope glycoprotein
synthesized in
hAdV/TRS/synORFS-infected 293 rtTA cells (Figure 6) or MARL-145 cells (data
not shown)
was 6 to 20 times the amount of the same protein synthesized in
hAdV/TR5/wtORFS infected
S1
CA 02418780 2003-02-28
cells. In Figure 6, left panel, hAdV/'TRS/synORFS-infected 293 rtTA cells
synthesized 11 times
more GPS than hAdV/TRS/wtORFS-infected cells, whereas in Figure 6, right
panel, the amount
of the GPS synthesized in hAdV/TRS/synORFS-infected cells was 6 times the
amount
synthesized in hAdV/TR5/wtORFS-infected cells, considering that the WT lane
was loaded with
3 times more cell lysates (in cpm) than the SYN lane. The ratio of lysate cpm
loaded in each
lane differed and is shown at the bottom of each panel in Figure 6.
Antibody response in pigs immunized with hAd UlTRSlwtORFS or hAdYlTRSlsynORFS
Following two intradermal injections of the control or test vaccine mixtures,
and before exposure
to PRRSV, none of the immunized piglets developed significant antibody titres
as revealed either
by indirect immunofluorescence (significant titres >16) on PRRSV-infected-MARC-
145 cells or
the virus neutralization assay (VN) (significant titres > 8). Furthermore,
reactivity to expression
of the authentic (wild type) GPS viral protein could not be demonstrated by
Western blotting
(Table 4, "Pre" data).
However, within 10 days of an intranasal viral challenge (Figure 7a and Table
4 "d10" data), the
three pigs pre-immunized with the test mixture of hAdV/TRS/synORFS and
AdCMV/tTA
developed significant antibody titres to the authentic viral GPS protein, as
demonstrated by IIF,
indirect ELISA, VN and Western blotting. Control pigs pre-immunized either
with
hAdV/TRS/wtORFS and AdCMV/tTA, or with the AdCMV/tTA vector alone displayed
antibody titres similar to synORFS-immunized pigs as detected by IIF and
indirect ELISA, but
did not develop significant VN antibody titres 10 days post-challenge. In the
case of
hAdV/TR5/wtORFS a weak reaction to the GPS protein was only demonstrated by
Western
blotting at 21 days post-challenge, while the pigs immunized with
hAdV/TR5/synORFS
developed a high specific immune response against the GP5 protein over the
same period (Figure
7b, Table 4 "d21" data). Thus only the pigs pre-immunized with the codon-
optimized synORFS
DNA rapidly developed significant VN antibody titres following viral
challenge. The data for
each pig are shown on a separate row in Table 4.
52
CA 02418780 2003-02-28
EXAMPLE 2: Codon-optimized ORF polynucleotides encoding ORF fragments
Materials and Methods
Construction of adenovirus-based fragment librariesl2,
Synthetic ORF polynucleotides encoding fragments (4synORFS) of IAF-Klop ORFS
protein are
being made by the exonuclease III technique' using a pCR2.l/synORFS
recombinant vector. The
synthetic ORFS is first cloned into the transfer vector pAdCMVS-P2DC-
GFPq/K7PSmlp
(obtained from Dr B. Massie). This vector permits the expression of both a
synthetic ORFS gene
and a GFPq (green fluorescence protein) reporter gene from a dicistronic mRNA.
The synthetic
ORFS is under the control of the CMV cumate promotor," which inhibits the
expression of toxic
truncated proteins in cells that constitutively express the cumate repressor,
CymR. The transfer
vector also possesses the protease gene (PS), which is essential for the
formation of viral
infectious particles. The PS gene provided by the transfer vector complements
the adenovirus
deleted for the PS gene. Consequently, 100%a of the adenoviruses recovered
from infected (with
adenovirus deleted for El and PS genes) and transfected (with the recombinant
transfer vector)
293 CymR cells (cells that constituvely express the eumate repressor for the
inhibition of the
transgene and complement the E1 deleted adenovirus) are 100% recombinant'2.
Importantly, this
new generation of replication competent adenoviruses do not require co-
infection of cells with an
adenovirus expressing a transactivator (AdCMV/tTA) for the expression of the
transgene. These
truncated translation products of these fragments will be tested for
expression, efficacy,
cytotoxicity, and/or for their ability to interact with other PPRSV proteins
or fragments, to select
those sequences most useful in the invention.
Between amino acid positions 26 to 39, some North American strains of PRRSV
ORFS protein
have no N-glycosylation site while others have three N-glycosylation sites
(positions 30, 33 and
34). SynORFS encodes the wild-type ORFS protein and possesses three N-
glycosylation sites
(positions 30, 44 and 51) of which two (44 and 51) are highly conserved in the
wild-type
protein3'. Direct mutagenesis will be used to replace the N asparagine residue
at positions 30, 44
and 51 simultaneously or independently in the full-length synORFS
polynucleotide and in
truncated variants (hAdV/~synORFS/ON). These N-mutant constructs will be
tested for
53
CA 02418780 2003-02-28
expression, efficacy, cytotoxicity, and/or for their ability to interact with
other PPRSV proteins
or fragments, to select those sequences most useful in the invention.
Expression of the truncated and N mutated synthetic ORFS recombinant
adenoviruses.
The transient expression of truncated ORFS proteins expressed by the pAdCMVS-
P2DC-
GFPq/K7PSmlp/OsynORFS will be tested as previously described. Western blotting
experiments can be done as previously described except that the new generation
of recombinant
adenoviruses do not need a co-infection with another adenovirus expressing a
transgene.
Toxicity of the truncated and N mutated synthetic ORFS recombinant
adenoviruses.
The deleted synthetic ORFS recombinant adenoviruses (hAdV/OsynORFS) will be
used to
evaluate which part of the protein is toxic and able to induced apoptosis. As
described above,
monolayers of non-helper cells (where the hAdV/~synORF5 could not replicate),
such as
MARC-145 cells (permissive cell line to PRRSV), will be infected at a MOI of
100 plaque
forming unit (PFU) per cell's and the toxicity will be evaluated by different
techniques as
previously described'S. Alternatively or in addition, alveolar macrophages
(the primary infected
cells in swine4~ 'o, zz. aa. as) will be infected in order to evaluate the
cytotoxicity and apoptosis
associated with deletion variants of the ORFS protein fragments. Abnormal
proliferation or
gross cellular changes that occur upon intracellular synthesis of the
truncated and N-mutated
ORFS peptides will be visualised under light microscope (Leyca, Leitz) at
various times up to 72
hours post-infection'S~'~. MARL-145 cells are not permissive to replication
defective (E1
deleted) recombinant hAdVs since they do not complement their E1 gene
functions, so any
cellular degeneration or abnormalities observed can be attributed to the
toxicity of the expression
of the PRRSV ORF5 peptide variants encoded by the synORFS variants. Two
techniques will be
used to characterize the cytopathic effect observe in hAdV-infected cells: the
fluorescence
TUNEL assay and the caspase 3 activation3;. The TUNEL assay detects the
fragmentation of
DNA which is characteristic of apoptosis. A commercial TUNEL assay (In Situ
cell death
detection kit, fluorescein, Roche, Laval, Quebec, Canada) will be used for the
detection of DNA
fragmentation in PRRSV and hAdVs-infected cells using the procedure
recommended by the
manufacturer. For the detection of caspase 3 activation, MARC-145 cells
infected with
hAdV/OsynORFS's wilt be disrupted at different times post-infection (p.i.) in
the lysis solution
provided in the ApoAlert Caspase~ Fluorescent Assay Kit (BD Biosciences
Clonetech, Palo
54
CA 02418780 2003-02-28
Alto, CA). Five pl (typically corresponding to 60 to 75 pg of protein) of the
cell lysates will be
added to 90 pl of a solution containing SO mM HEPES, pH 7.0, 10% glycerol,
0.1% CHAPS,
2mM EDTA and 5mM dithiothreitol (DTT). 10 pM of the DEVD-AFC fluorogenic
substrate
specific for caspase 3, (Biomol Research Laboratories Inc., Plymouth Meeting,
PA), will then be
added and the rate of fluorescence released monitored using a 96-well plate
fluorimeter
(Cytofluor, Perseptive Biosystems, Foster City, CA). The results are expressed
as fluorescence
released (fluorescence unit or FU) per sec per pg of cell lysate.
Epitopes of synORFS implicated in the viral neutralization phenomenon.
Monocloanl antibodies (MoAbs) capable of neutralizing the viral infectivity in
MARC-145 cells
of the IAF-Klop strain of PRRSV are known in the art29~ 3' and can be used in
the virus
neutralization test (VN) described above. Other MoAbs directed specifically
against the GPS
protein are being produced and characterized using standard techniques known
in the art. Those
with the appropriate anti-PRRSV activity can also be used in the VN test.
These antibodies can also be used in IIF assays as follows: Monolayers of
continuous cell lines,
such as MARC-145 cells, and 293 cells, are infected with synORF
polynucleotides expressing
fragments or variants. At different time p.i., cell will be fixed with a cold
80% acetone solutiong~
'4. An indirect immunofluorescence test (IIF, previously described) performed
with an array of
neutralizing MoAbs will establish their specificity against each of the
epitopes of the GPS
protein. A positive result with a particular ORF variant will help identify a
neutralizing epitope
of the GPS protein, which can be exploited in the design of further candidate
ORF constructs with
improved efficacy.
Immunization of animals with the truncated and N mutated synthetic ORFS
polynucleotides
The immunization procedure and follow up assays to evaluate the immune
response induced in
immunized pigs are described above (VN, IIF, ELISA, necropsy,
histopathological examination,
western blot, blastogenic transformation test, virus isolation).
EXAMPLE 3: synORF4 and synORF6
While the major glycosylated envelope protein GP5''~ m. z7-3o. 32. 36 plays a
major role in inducing
neutralizing antibodies and a protective immune response in pigs, other
proteins also likely to
contribute to the immune response include the major structural protein M 2"''
S encoded by ORF6,
and the minor structural protein GP4'~ encoded by ORF4.
CA 02418780 2003-02-28
It as been demonstrated that for a related virus, the equine arteritis virus
(EAV), that an improved
immune response is obtained when animals (mice and horse) are immunized with a
recombinant
alphavirus vaccine that expresses the heterodimer GP5-M versus those that
express the GPS
alonez~'. Also the highest cellular immune response in pigs is specifically
directed against the M
S proteins. Thus it is contemplated that expression of the M protein or
fragments thereof may be
useful in the development of an efficient vaccine against PRRSV.
The glycosylated minor structural protein GP4, encoded by ORF4 of PRRSV, may
also play a
role in the induction of a protective immune response. The GP4 of a European
strain of PRRSV
was able to induce an appreciable level of neutralizing antibodies26 however
only very low levels
of neutralizing antibodies could be detected upon immunization with the GP4 of
a North
American PRRSV strain2' which is 68% identical at the amino acid level. The
poor expression
of the ORF4 gene in PRRSV infected cells and by recombinant vectorszs and low
quantity of
purified infectious viral particles, may explain why experimentally infected
pigs did not develop
significant level of neutralizing antibodies". A completely optimized full-
length synORF4
polynucleotide sequence was designed based on the wild type ORF4 of IAF-Klop
strain of
PRRSV.
Materials and Methods
The procedure used to construct the synthetic ORF4 and ORF6 of PRRSV is
described above.
Figures 10 and 11 show nucleotide sequences of the optimized synORF4 and
synORF6 nucleic
acid molecules. The nucleotide identities of synORF4 and synORF6 compared to
their wild-type
genes are 78% and 79% respectively. In each case, preferred codons have been
substituted for
104 non-preferred codons without altering the deduced amino acid sequence. 58%
and 59% of
the codons have been replaced in the sequence of synORF4 and synORF6,
respectively. Table
3b and Table 3c show the proportion of codons used to encode each amino acid
in the wild-type
sequences of the IAF-Klop strain of PRRSV, compared with the codons used in
human cells.
EXAMPLE 4: Partial codon-o timization of ORFS
Figure 8 and SEQ ID NO: 42 show the nucleotide sequence of the partially
optimized synORFS
variant and the modified amino acid sequence it encodes is provided in SEQ ID
NO: 43. This
polypeptide differs from the wild-type ORF_5 protein sequence at 4 positions:
at amino acid
position 48, Y replaces C, at amino acid position 63, S replaces A, at amino
acid position 155 L
replaces W, and at amino acid position 183, A replaces G. The altered
nucleotides underlying
56
CA 02418780 2003-02-28
these amino acid substitiutions are shown boxed in Figure 8. The identity of
the polypeptide
expressed from the synORFS variant is 98% compared to the polynucleotide of
the wild-type
(and completely optimized synthetic). As shown in Figure 8, a total of 14
nucleotide are different
in the synORFS variant compared to the synORFS, corresponding to a nucleotide
identity of
97%.
The wild-type ORF5 and the synORFS variant were cloned into the plasmid vector
pVAX
(Invitrogen Canada Inc, Burlington, Ontario). 293 cells were transfected with
these recombinant
vectors, and a pVAX control vector, as previously described. Expression of the
GP5 protein was
monitored using the IIF technique previously described. Figure 9 demonstrates
that an increased
expression level was also obtained with the partially optimized synORFS
variant.
The invention being thus described, it will be obvious that the same may be
varied in many ways.
Such variations are not to be regarded as a departure from the spirit and
scope of the invention,
and all such modifications as would be obvious to one skilled in the art are
intended to be
included within the scope of the following claims. Patents and publications
referred to
throughout this application are hereby incorporated by reference in their
entirety.
57
CA 02418780 2003-02-28
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activation of monocytes/macrophages on their susceptibility to porcine
reproductive and respiratory
syndrome virus (PRRSV). Arch Viml 142:2483-97.
11 Eaton, R. W. 1997. p-Cymene catabolic pathway in Pseudomonas putida Fl:
cloning and characterization
of DNA encoding conversion of p-cymene to p-cumate. JBacteriol 179:3171-80.
12 Elahi, S. M., W. Oualikene, L. Naghdi, et aL 2002. Adenovirus-based
libraries: efficient generation of
recombinant adenoviruses by positive selection with the adenovirus protease.
Gene Ther 9:1238-46.
13 Fernandez, A., P. Suarez, J. M. Castr~ et al. 2002. Characterization of
regions in the GP5 protein of
porcine reproductive and respiratory syndrome virus required to induce
apoptotic cell death. Virus Res
83:103-18.
14 Gagnon. C. A. and S. Dea. 1998. Differentiation between porcine
reproductive and respiratory syndrome
virus isolates by restriction fragment length polymorphism of their ORFs 6 and
7 genes. Can J Vet Res
b2:1 I U-6.
58
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15 Gagnon, C. A., G. Lachapelle, Y. Langelier, et al. 2003. Adenoviral-
expressed GP5 is distinct from the
authentic protein that is included in the porcine reproductive and respiratory
syndrome virions. Arch
Virol:In press.
16 Gagnon, C. A., Y. Langelier, B. Massie, et al. 2001. Biochemical properties
and processing of the three
major structural proteins of PRRS virus expressed by recombinant adenoviruses.
Structural, functional and
community aspects. Adv Exp Med Biol 494:225-31.
17 Gonin, P., B. Pirzadeh, C. A. Gagnorry et al. 1999. Seroneutralization of
porcine reproductive and
respiratory syndrome virus correlates with antibody response to the GP5 major
envelope glycoprotein. J
Vet Diagn Invest 11:20-6.
18 Halbur, P. G., P. S. Paul, M. L. Frey, et al. 1996. Comparison of the
antigen distribution of two US
porcine reproductive and respiratory syndrome virus isolates with that of the
Lelystad virus. Vet Pathol
33: I 59-70.
19 Halbur, P. G., P. S. Paul, M. L. Frey, e1 n1. 1995. Comparison of the
pathogenicity of two US porcine
reproductive and respiratory syndrome virus isolates with that of the Lelystad
virus. Vet Pathol 32:648-60.
20 Halbur, P. G., P. S. Paul and X. J. Meng. 1994. Marked variability in
pathogenicity of nine US porcine
reproductive and respiratory syndrome virus (PRRSV) isolates in 5-weeks-old
CDCD pigs. Proceedings of
the 13th International Pig Veterinary Congress:59.
21 Kwang, J., F. Zuckermann, G. Ross, et al. 1999. Antibody and cellular
immune responses of swine
following immunisation with plasmid DNA encoding the PRRS virus ORF's 4, 5. 6
and 7. Res Vet Sci
67:199-ZO 1.
22 Larochelle, R., H. Mardassi, S. Dea, et al. 1996. Detection of porcine
reproductive and respiratory
syndrome virus in cell cultures and formalin-fixed tissues by in situ
hybridization using a digoxigenin-
labeled probe. J Vet Diagrr Invest 8:3-10.
23 Massie, B., F. Couture, L. Lamoureux, et al. 1998. Inducible overexpression
of a toxic protein by an
adenovirus vector with a tetracycline-regulatable expression cassette. J Virol
72:2289-96.
24 Meng, X. J., P. S. Paul, P. G. Halbur, et al. 1996. Characterization of a
high-virulence US isolate of
porcine reproductive and respiratory syndrome virus in a continuous cell line,
ATCC CRL11171. J Vet
Diagn Invest 8:374-81.
25 Meulenberg. J. J., A. Petersen-den Besten, E. P. De Kluyver, et al. 1995.
Characterization of proteins
encoded by ORFs 2 to 7 of Lelystad virus. Virology 206:155-63.
26 Meulenberg, J. J., A. P. van Nieuwstadt, A. van Essen-Zandbergen, et al.
1997. Posttranslational
processing and identification of a neutralization domain of the GP4 protein
encoded by ORF4 of Lelystad
virus. J Virol 71:6061-7.
27 Osorio, F. A., J. A. Galeota, E. Nelson, et al. 2002. Passive transfer of
virus-specific antibodies confers
protection against reproductive failure induced by a virulent strain of
porcine reproductive and respiratory
syndrome virus and establishes sterilizing immunity. Virology 302:9-20.
28 Ostrowski, M., J. A. Galeota, A. M. Jar, et al. 2002. Identification of
neutralizing and nonneutralizing
epitopes in the porcine reproductive and respiratory syndrome virus GPS
ectodomain. J Virol76:4241-.50.
29 Pirzadeh, B. and S. Dea. 1997. Monoclonal antibodies to the ORFS product of
porcine reproductive and
respiratory syndrome virus define linear neutralizing determinants. JGen Virol
78:1867-73.
30 Pirzadeh, B. and S. Dea. 1998. Immune response in pigs vaccinated with
plasmid DNA encoding ORFS
of porcine reproductive and respiratory syndrome virus..l Gen Virnl 79:989-99.
59
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31 Pirzadeh, B., C. A. Gagnon and S. Dea. 1998. Genomic and antigenic
variations of porcine reproductive
and respiratory syndrome virus major envelope GPS glycoprotein. Can J Vet Res
62:170-7.
32 Plagemann, P. G., R. R. Rowland and K. S. Faaberg. 2002. The primary
neutralization epitope of
porcine respiratory and reproductive syndrome virus strain VR-2332 is located
in the middle of the GPS
ectodomain. Arch Virol 147:2327-47.
33 Thornberry, N. A. and Y. Lazebnik. 1998. Caspases: enemies within. Science
281:1312-6.
34 Voicu, I. L., A. Silim, M. Morin, et al. 1994. Interaction of porcine
reproductive and respiratory
syndrome virus with swine monocytes. Vet Rec 134:422-3.
35 Wensvoort, G., C. Terpstra, J. M. Pol, et al. 1991. Mystery swine disease
in The Netherlands: the
isolation of Lelystad virus. Vet Q 13:121-30.
36 Zhang, Y., R. D. Sharma and P. S. Paul. 1998. Monoclonal antibodies against
conformationally
dependent epitopes on porcine reproductive and respiratory syndrome virus. Ver
Microbiol 63:125-36.
CA 02418780 2003-02-28
Table 1. North American PRRSV strains and their GenBank accession numbers.
PRRSV Strain AccessionReference
tar-atop t~uenec U64928 Mardassi et u1., (1994) Can. J.
reference cytopathic Vet. Res. 58:55-64
strain)
IAF-DESR U64930 Pirzadeh et al., (1998) Can J Vet
Res 62:170-177
IAF-BAJ U64929 Pirzadeh et al., (1998) Can J Vet
IAF-CM AF013106 Res 62:170-177
Pirzadeh et al., (1998) Can J Vet
Res 62:170-177
IAF-93-653 U64931 Pirzadeh et al. , (1998) Can J Vet
IAF-94-3182 U64933 Res 62:170-177
Pirzadeh et al., (1998) Can J Vet
Res 62:170-177
IAF-94-287 U64934 Pirzadeh etal., (1998) Can J Vet
Res 62:170-177
IAF-93-2616 U64932 Pirzadeh etal., (1998) Can J Vet
Res 62:170-177
ONT-TS U64935 Pirzadeh et al., (1998) Can J Vet
Res 62:170-177
tso ate Magar et al. , (1995) Can J Vet
US isolate 91-22778 Res 59:232-234
US isolate 91-1453
ATCC VR-2332 U87392 Nelsen et al., (1999) J Virol 73:270-280
(US reference strain)
ATCC VR-2332 ~1~~4- NieIseo et al.~ (2003) J Virol 77:
(US reference strain) in press
ATCC VR-2385 U03040 Meng et al., (1994) Arch Virol 75:1795-1801
(US reference strain)
ISUVDL 98-38803 AF535152 Opriessnig et al., (2002) J Virol
76:11837-11844
61
CA 02418780 2003-02-28
Table 2. Examples of preferred codons for optimal expression in mammalian
cells
AMINO ACID POSSIBLE CODONS PREFERRED CODON
Alanine GCA GCT GCC
Arginine CGA AGG CGC
CGT AGA
-
. _
-. _
sparagme ~~ AA~
_
spartic ci ~ _
ysteme _
fig.
utamme
utamic ci AAA
Glycine GGA GGT GGC
Histidme ~H~ ~H i
Isoleucine ATT ~ ATC
Leucine CTA TTG CTG
CTT TTA
ysme HH~ rah
Proline CCA CCT CCC
- - _ _ _
eny a amne
Serine TCA AGT TCC
TCT AGC
Threonine ACA ACT ACC
___ _ _
_
yrosme ~ TAC
Valine GTT GTC I GTG
62
CA 02418780 2003-02-28
Table 3a. Frequency of codon occurrence in humans (H) and in ORFS of PRRSV '
nucleotide# H PRRSV nucleotide# H PRRSV nucleotide# H PRRSV
GPS GPS GPS
AA I % % (#) AA 1 % % (#) AA 1 & % % (#)
& & 2 3
2 2
3 3
Ala GC C 53 27 (4) Gly GG C 50 33 (4) Phe TT C 80 33
(3)
T 17 20 (3) T 12 33 (4) T 20 67
(6)
A 13 20 (3) A 14 8 (1)
G 17 33 (5) G 24 25 (3) Ser TC C 28 26
(5)
T 13 5 (1)
Arg CG C 37 12,5 His CA C 79 50 (2) A 5 16 (3)
(1)
T 8 25 (2) T 21 SO (2) G 9 10,5
(2)
A 6 0 (0) AG C 34 37
(7)
G 21 25 (2) Ile AT C 77 45 (5) T 10 5 (1)
AG A 10 25 (2) T 10 36 (4)
G 18 12,5 A 5 18 (2) Thr AC C 57 57
(1) (8)
T 14 21
(3)
Leu CT C 28 17 (4) A 14 7 (
1 )
Asn AA C 78 57 (4) T 5 13 (3) G 15 14
(2)
T 22 43 (3) A 3 0 (0)
G 58 30 (7) Tyr TA C 74 33
(3)
Asp GA C 75 43 (3) TT A 2 0 (0) T 26 67
(6)
T 25 57 (4) G 8 39 (9)
Val GT C 25 42
(8)
Cys TG C 68 45 (5) Lys AA A 18 78 (7) T 7 26 (5)
T 32 55 (6) G 82 22 (2) A 5 5 (1)
G 64 26
(5)
Gln CA A 12 75 (3) Pro CC C 48 33 (2)
G 88 25 (1) T 19 50 (3)
A 16 0 (0)
Glu GA A 25 20 (1) G 17 17 (1)
G 75 80 (4)
' The frequencies of the individual codons are shown as percentages for each
of the degenerately encoded amino
acids, as well as the number of each amino acid for ORF (in parentheses). The
nu>st prevalent codon is shown
underlined in bold. The ORF protein is from the IAF-Klop strain of PRRSV.
63
CA 02418780 2003-02-28
Table 3b. Frequency of codon occurrence in humans (H) and in ORF4 of PRRSV
H GP4 H GP4 H GP4
AlaC 53 31 (4) A 25 25 (Z) Pro C 4$-
GLU
GCT 17 15 (2) GA G 75 75 (6) CC T 19 SO (2)
A 13 23 (3) A 16 25 (1)
G 17 31 (4) Gly - G 17 0 (0)
GG T 12 17 (1)
A 14 17 (1) Phe
Arg- G 24 17 ( TT T 20 36 (4)
1 )
CGT 8 14 (1)
A 6 14 (1) His Ser _
G 21 0 (0) CA T 21 80 (4) TC T 9 36 (8)
A 8 9 (2)
AGA 10 0 (0) Ite G 19 9 (2)
G 18 29 (2) AT T 17 33 (4)
A 6 2S (3) AG C 10 9 (2)
T 17 18 (4)
Asn~ 7~~~ Leu
AAT 22 60 (3) CT T 5 33 (7) Thr
A 3 0 (0) AC T 14 7 (1)
G 59 10 (2) A 14 27 (4)
G 1 S 20 (3)
Asp- TT A 2 10 (2)
GAT 32 20 (1) G .5 19 (4) Tyr
TA T 26 60 (3)
Cys- Lys
TGT 32 57 (4) AA G 82 60 (3) Val
GT T 7 31 (5)
A 5 6 (1)
Gln G 63 31 (5)
CAG 88 40 (2)
64
CA 02418780 2003-02-28
Table 3c. Frequency of codon occurrence in humans (H) and in ORF6 of PRRSV
H M H M H M
-
Ala~ ~ 39 i~ A Pro
GLU
GC T 17 11 (2) GA G 75 50 (1) CC T 19 17 (1)
A 13 22 (4) A 16 33 (2)
G 17 28 (5) Gly - G 17 17 (1)
GG T 12 8 (1)
A 14 15 (2) Phe
Arg~ _ G 24 31 (4) TT T 20 40 (4)
CG T 8 22 (2)
A 6 11 (1) His Ser _
G 21 22 (2) CA T 21 17 (1) TC T 9 8 (1)
A 8 8 (1)
AG A 10 22 (2) Ile G 19 8 (1)
G 18 0 (0) AT T 17 43 (3)
A 6 29 (2) AG C 10 17 (2)
T 17 33 (4)
Asn~ Leu
AA T 22 50 (3) CT T .5 13 ( 3) Thr
A 3 17 (4) AC T 14 17 (2)
G 59 22 (5) A 14 2S (3)
G 15 25 (3)
- -
-
AspC TT A 2 4 (1 )
61~
75Z3j
GA T 32 25 (1) G 5 22 (5) Tyr
TA T 26 50 (3)
-
CysC Lys
-
TG T 32 25 (1) AA G 82 30 (3) Val
GT T 7 22 (4)
A 5 28 (5)
Gln G 63 28 (5)
CA G 88 50 (I)
CA 02418780 2003-02-28
Table 4. Post-challenge antibody response to immunization with DNA vaccines
expressing either
the wild type or synthetic codon-optimized PRRSV ORES gene.
Sero- Immuno-
Western blotneutralisation' fluorescencez IDEXX
Ratio
Antibody Antibody (ELISA)
titres titres
VirusesPre d10 d21 Pre d10 Pre d10 d21 Pre d10 d21
- - 16 < 64 >1024 0 0,489 1,243
16
Ad /tTA- - - < >256 >1024 0 1,050 1,401
16 <512
12 < 256 >1024 0 0,772 1,478
16
Ad/syn + + 256 < 128 >1024 0 1,239 1,443
16
ORFS
w w + 128 < >64 <128>1024 0 0,673 1,211
16
+
Ad/tTA + + 256 < 64 >1024 0 0,700 1,529
16
Ad/wt w ~'~' - < >16 <64 >1024 0 0,605 1,281
16
ORFS
- + 8 < >16 <64 >1024 0 0,730 1,509
16
Ad/tTA - ~'~' 8-16 < 16 >1024 0 0,480 1,265
16
Seroneutralisation antibody titres are expressed as the reciprocal of highest
serum dilution which inhibited
cytopathic changes produced by 100 TCIDSO of the IAF-Klop Strain of PRRSV in
MARC-145 cells.
Z Immunofluorescence antibody titres are expressed as the reciprocal of the
highest serum dilution at which specific
fluorescence was observed in PRRSV-infected cells.
66
CA 02418780 2003-02-28
Table 5. Oligonucleotide primers used in PCR amplification of the synthetic
ORFS.
Name SEQ Sense Positions" SEQUENCEb
ID
NO:
1 50 + -9 to 60 5' accggatccA
T
GCTGGGCAAGTGCCTGACCGCCGGCTGT
_ 3'
_
TGCTCCCAGCTGCCCTTCCTGTGGTGTATC
2 51 + 61 to 120 5' GTGCCCTTCTGTTTCGCCGCCCTGGTGAAC
GCCTCCTCCTCC'TCCTCCTCCCAGCTGCAG3'
3 52 + 121 to 180 15'TCCATCTACAACCTGACCATCTGTGAGCTG
AACGGCAC'.CGACTGGCTGAACAAGAACTTC3'
4 53 + 181 to 240 5' GACTGGGCCGTGGAGACCTTCGTGATCTTC
('CCGTGCTGACCCACATCGTGTCCTACGGC3'
54 + 241 to 300 5' C;CCCTGACCACCTCCCACTTCC'PGGACGCC
GTGGGCCI'GATCACCGTGTCCACCGCCGGC3'
6 55 + 301 to 360 5' TACTACCACGGCCGCTACGTGCTGTCCTCC
C;TGTACGCCGTGTGCGCCCTGGCCGCCCTG3'
7 56 + 361 to 420 5' ATCTGCTTCGTGATCCGCCTGACCAAGAAC
TGCATGTCCTC;GCGCTACTCCTGTACCCGC3'
8 57 + 421 to 980 5' TACACCAACTTCC'"GCTGGACTCCAAGGGC
AAGCTGTACCGCTGGCGCTCCCCCGTGATC3'
9 58 + 481 to 540 5' ATCGAGAAGGGCGGCAAGGTGGAGGTGGAC
GGCCACCTGATCGACCTGAAGCGCGTGGTG3'
59 + 541 to 600 5' CTGGACGGCTCCG(_'CGCC.'ACCCCCGTGACC
AAGGTGTCCGCCGAGCAGTGGTGTCGCCCC3'
11RC 60 - 30 to 1 5' ACAGCCGGCGGTCAGGCACTTGCCCAGCAT3'
1RC 61 - 90 to 31 5' GTTCACC:AGGGCGGCGAAACAGAAGGGCAC
GATACACCACAGGAAGGGCAGCTGGGAGCA3'
2RC 62 - 150 to 91 5' C:AGCTCACAGATGGTCAGGTTGTAGATGGA
~
CTGCAGCTGGGAGGAGGAGGAGGAGGAGGC3'
3RC 63 - 210 to 151 5' GAAGATCACGAAGGTCTCCACGGCCCAGTC
__ C;AAGTTC,'T'rGT'TC~'~GCC.'AGTCGGTGCCGTT3'
4RC 64 - 270 to 211 S' GGCGTCCAGGAAGTGG<~F.GGTGGTCAGGGC
'
' GCCGTAGGACACGATGTGGGTCAGCACGGG3'
5RC 65 - 330 to 2 S' GGAGGACAGCACGTAGC.'GGCCGTGGTAGTA
71
~'
GCCGGCGGTGGAC~'1CGC.Z'(,ATCAGGCCCAC3'
6RC 66 - 390 to 331 5' GTTCTTGGTCAGG!'GGA'C(=ACGAAGCAGAT
~i
C.'AGGG(.',GGCCAGGGCC;C'ACACGGCGTACAC3'
7RC 67 - 450 to 391 5' GCCCTTGGAGTCCAGCAGGAAGTTGGTGTA
;
GCGGGTACAGGAGTAGCGCCAGGACATGCA3'
8RC 68 - 510 to _ S' GTCCACCTCCACC'rTGC'CGCCCTTCTCGAT
451
'
__ GATCACGGGi,GAGCGCC'AGCGGTACAGCTT3'
9RC 69 - 570 to 511 5' GGTCAC.GGGGGTGGCGGCGGAGCCGTCCAG
CACCACGCGCTTCAGGTC'GATCAGGTGGCC3'
lORC 70 - 611 to 5 5' ggccrgatcCT
7 AGGGGCC;ACACCACTGCTCG
I
_
(~CGGACACC'1'.C 3'
a Positions are indicated according to the predesigned entire synthetic ORFS
sequence, A in the initiation codon
being the +1 position.
° Upper cases refer to synthetic ORFS specific nucleotides. Lower cases
refer to non-ORFS sequence-extra
nucleotides containing BamHI restriction site. Start and stop codons are
underlined.
67
CA 02418780 2003-02-28
Sequence Listing
(1) GENERAL INFORMATION:
(I)APPLICANT: Institut national de la Recherche Scientific
Dea, Serge
(ii) TITLE OF INVENTION: Synthetic Genes Encoding Proteins Of Porcine
Reproductive And Respiratory Syndrome Virus And Use Thereof
(iii) NUMBER OF SEQUENCES: 72
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: MBM & C0.
(B) STREET: P.O. BOX 809, STATION B
(C) CITY: OTTAWA
(D) PROVINCE: ONTARIO
(E) COUNTRY: CANADA
(F) POSTAL CODE: K1P 5P9
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: Windows
(D) SOFTWARE: Word
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: N/A
(B) FILING DATE: February 28, 2002
(C) CLASSIFICATION.
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: SWAIN, Margaret
(B) REGISTRATION NUMBER: 10926
(C) REFERENCE/DOCKET NUMBER: 255-130
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 613/567-0762
(B) TELEFAX: 613/563-7671
(2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 603 base pairs
(B) TYPE: Nucleic acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(ii) MOLECULE TYPE: DNA
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Artificial Sequence
(ix) FEATURE:
(A) NAME/KEY:CDS
(B) LOCATION: (1)...(603)
(D) OTHER INFORMATION: synORF5 encoding GP5 of PRRSV strain
IAF-Klop
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
1
CA 02418780 2003-02-28
ATGCTGGGC TGC CTGACCGCC GGCTGTTGC TTCCAGCTG CCCTTC 48
AAG
MetLeuGlyLysCys LeuThrAla GlyCysCys PheGlnLeu ProPhe
1 5 10 15
CTGTGGTGTATCGTG CCCTTCTGT TTCGCCGCC CTGGTGAAC GCCTCC 96
LeuTrpCysIleVal ProPheCys PheAlaAla LeuValAsn AlaSer
20 25 30
TCCTCCTCCTCCTCC CAGCTGCAG TCCATCTAC AACCTGACC ATCTGT 144
SerSerSerSerSer GlnLeuGln SerIleTyr AsnLeuThr IleCys
35 40 45
GAGCTGAACGGCACC GACTGGCTG AACAAGAAC TTCGACTGG CCCGTG 192
GluLeuAsnGlyThr AspTrpLeu AsnLysAsn PheAspTrp ProVal
50 55 60
GAGACCTTCGTGATC TTCCCCGTG CTCACCCAC ATCGTGTCC TACGGC 240
GluThrPheValIle PheProVal LeuThrHis IleValSer TyrGly
65 70 75 80
GCCCTGACCACCTCC CACTTCCTG GACGCCGTG GGCCTGATC ACCGTG 288
AlaLeuThrThrSer HisPheLeu AspAlaVal GlyLeuIle ThrVal
85 90 95
TCCACCGCCGGCTAC TACCACGGC CCCTACGTG CTGTCCTCC GTGTAC 336
SerThrAlaGlyTyr TyrHisGly ProTyrVal LeuSerSer ValTyr
100 105 110
GCCGTGTGCGCCCTC GCCGCCCTG ATTTGCTTC GTGATCCGC CTGACC 384
AlaValCysAlaLeu AlaAlaLeu IleCysPhe ValIleArg LeuThr
115 120 125
AAGAACTGCATGTCC TGGCGCTAC TCCTGTACC CGCTACACC AACTTC 432
LysAsnCysMetSer TrpArgTyr SerCysThr ArgTyrThr AsnPhe
130 135 140
CTGCTGGACTCCAAG GGCAAGCTG TACCGCTGG CGCTCCCCC GTGATC 480
LeuLeuAspSerLys GlyLysLeu TyrArgTrp ArgSerPro ValIle
145 150 155 160
ATCGAGAAGGGCGGC AAGGTGGAG GTGGACGGC CACCTGATC GACCTG 528
IleGluLysGlyGly LysValGlu ValAspGly HisLeuIle AspLeu
165 170 175
AAGCGCGTGGTGCTC GACGGCTCC GCCGCCACC CCCGTGACC AAGGTG 576
LysArgValValLeu AspGlySer AlaAlaThr ProValThr LysVal
180 185 190
TCCGCCGAGCAGTGG TGTCGCCCC TAG 603
SerAlaGluGlnTrp CysArgPro
I95 200
2)
INFORMATION
FOR
SEQ
ID
N0:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH:200 amino acids
(B) TYPE: Amino Acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Protein
2
CA 02418780 2003-02-28
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Artificial Sequence
(ix) FEATURE:
(A) NAME/KEY:
(B) LOCATION:
(D) OTHER INFORMATION:SynORFS of PRRSV strain IAF-Klop
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:2:
Met Leu Gly Lys Cys Leu Thr Ala Gly Cys Cys Phe Gln Leu Pro Phe
1 5 IO 15
Leu Trp Cys Ile Val Pro Phe Cys Phe Ala Ala Leu Val Asn Ala Ser
20 25 30
Ser Ser Ser Ser Ser Gln Leu Gln Ser Ile Tyr Asn Leu Thr Ile Cys
35 40 45
Glu Leu Asn Gly Thr Asp Trp Leu Asn Lys Asn Phe Asp Trp Pro Val
50 55 60
Glu Thr Phe Val Ile Phe Pro Val Leu Thr His Ile Val Ser Tyr Gly
65 70 75 80
Ala Leu Thr Thr Ser His Phe Leu Asp Ala Val Gly Leu Ile Thr Val
85 90 95
Ser Thr Ala Gly Tyr Tyr His Gly Pro Tyr Val Leu Ser Ser Val Tyr
100 105 110
Ala Val Cys Ala Leu Ala Ala Leu Ile Cys Phe Val Ile Arg Leu Thr
115 120 125
Lys Asn Cys Met Ser Trp Arg Tyr Ser Cys Thr Arg Tyr Thr Asn Phe
130 135 140
Leu Leu Asp Ser Lys Gly Lys Leu Tyr Arg Trp Arg Ser Pro Val Ile
145 150 155 160
Ile Glu Lys Gly Gly Lys Val Glu Val Asp Gly His Leu Ile Asp Leu
165 170 175
Lys Arg Val Val Leu Asp Gly Ser Ala Ala Thr Pro Val Thr Lys Val
180 185 190
Ser Ala Glu Gln Trp Cys Arg Pro
195 200
(2) INFORMATION FOR SEQ ID N0:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1563 base pairs
(B) TYPE: Nucleic acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(ii) MOLECULE TYPE: DNA
(vi) ORIGINAL SOURCE:
(A) ORGANISM:Sus scrofa
(ix) FEATURE:
(A) NAME/KEY:CDS
(B) LOCATION: (1)...(603)
(D) OTHER INFORMATION: wtORF5 encoding GP5 of PRRSV
strain IAF-Klop GENBank accession #U64928
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:3:
3
CA 02418780 2003-02-28
ATG TTG GGG AAA TGC TTG ACC GCG GGC TGT TGC TCG CAA TTG CCT TTT 48
Met Leu Gly Lys Cys Leu Thr Ala Gly Cys Cys Ser Gln Leu Pro Phe
1 5 10 15
TTG TGG TGT ATC GTG CCG TTC TGT TTT GCT GCG CTC GTC AAC GCC AGC 96
Leu Trp Cys Ile Val Pro Phe Cys Phe Ala Ala Leu Val Asn Ala Ser
20 25 30
AGC AGC AGC AGC TCC CAA TTG CAG TCG ATT TAT AAC CTG ACG ATA TGT 144
Ser Ser Ser Ser Ser Gln Leu Gln Ser Ile Tyr Asn Leu Thr Ile Cys
35 40 45
GAG CTG AAT GGC ACA GAT TGG CTG AAT AAA AAT TTT GAT TGG GCA GTG 192
Glu Leu Asn Gly Thr Asp Trp Leu Asn Lys Asn Phe Asp Trp Ala Val
50 55 60
GAG ACT TTT GTT ATC TTT CCT GTG TTG ACT CAC ATT GTC TCC TAT GGC 240
Glu Thr Phe Val Ile Phe Pro Val Leu Thr His Ile Val Ser Tyr Gly
65 70 75 80
GCC CTC ACC ACC AGC CAT TTC CTT GAC GCA GTC GGT CTG ATC ACT GTG 288
Ala Leu Thr Thr Ser His Phe Leu Asp Ala Val Gly Leu Ile Thr Val
85 90 95
TCT ACC GCC GGA TAT TAC CAC GGG CGG TAT GTC TTG AGT AGC GTC TAC 336
Ser Thr Ala Gly Tyr Tyr His Gly Arg Tyr Val Leu Ser Ser Val Tyr
100 105 110
GCT GTC TGC GCC TTG GCT GCG CTG ATT TGC TTC GTC ATT AGG TTG ACG 384
Ala Val Cys Ala Leu Ala Ala Leu Ile Cys Phe Val Ile Arg Leu Thr
115 120 125
AAA AAC TGC ATG TCC TGG CGC TAC TCA TGT ACC AGA TAT ACC AAC TTT 432
Lys Asn Cys Met Ser Trp Arg Tyr Ser Cys Thr Arg Tyr Thr Asn Phe
130 135 140
CTT CTG GAC TCC AAG GGC AAA CTC TAT CGT TGG CGG TCA CCC GTC ATC 480
Leu Leu Asp Ser Lys Gly Lys Leu Tyr Arg Trp Arg Ser Pro Val Ile
145 150 155 160
ATA GAG AAA GGG GGT AAA GTT GAG GTT GAT GGT CAT CTG ATC GAC CTC 528
Ile Glu Lys Gly Gly Lys Val Glu Val Asp Gly His Leu Ile Asp Leu
165 170 175
AAG AGA GTT GTG CTT GAT GGT TCC GCG GCA ACC CCT GTA ACC AAA GTT 576
Lys Arg Val Val Leu Asp Gly Ser Ala Ala Thr Pro Val Thr Lys Val
180 185 190
TCA GCG GAA CAA TGG TGT CGT CCC TAG ACGACTTCTG CAATGACAGC 623
Ser Ala Glu Gln Trp Cys Arg Pro
195 200
ACGGCCCCAC AAAAGGTACT CCTGGCGTTT TCTATCACCT ACACGCCAGT AATGATATAT 683
GCCCTAAAGG TAAGTCGCGG CCGACTGCTA GGGCTTCTGC ACCTTTTAAT TTTCCTGAAT 743
TGTGCTTTCA CCTTCGGGTA TATGACATTC GCGCACTTTC AGAGTACAAA TAAAGTCGCG 803
CTCACTATGG GAGCAGTAGT TGCGCTCCTT TGGGGGGTGT ACTCAGCCAT AGAAACCTGG 863
AAATTCATCA CCTCCAGATG CCGTTTGTGC TTGCTAGGCC GCAAGTACAT TCTGGCCCCT 923
GCCCACCACG TTGAGAGTGC CGCAGGCTTT CATCCGATTG CGGCAAGTGA TAACCACGCA 983
TTTGTCGTCC GGCGTCCCGG CTCCACTACG GTTAACGGCA CATTGGTGCC CGGGTTGAAA 1043
AGCCTCGTGT TGGGTGGCAG AAAAGCTGTC AAACGGGGAG TGGTAAACCT CGTTAAATAT 1103
4
CA 02418780 2003-02-28
GCCAAATAAC AACGGCAAGC AGCAGAAGAA AAAGAAAGGG GATGGCCAGC CAGTCAATCA 1163
GCTGTGCCAG ATGCTGGGCA GGATCATCGC CCAGCAAAAC CAGTCCAGAG GTAAGGGACC 1223
GGGGAAGAAA AATAAGAAGA AAAACCCGGA GAAGCCCCAT TTTCCTCTAG CGACTGAAGA 1283
TGACGTCAGA CATCACTTCA CCCCTAGTGA GCGGCAATTG TGTCTGTCGT CAATCCAGAC 1343
TGCCTTTAAT CAAGGCGCTG GAACTTGTAC CCTATCAGAT TCAGGGAGAA TAAGTTACGC 1403
TGTGGAGTTT AGTTTGCCTA CGCATCATAC TGTGCGCCTG ATTCGCGTCA CAGCATCACC 1463
CTCAGCATGA TGAGCTGGCA TTCTTGAGAC ATCCCAGTGT TTGAATTGGA AGAATGTGTG 1523
GTGAATGGCA CTGATTGATA TTGTGCCTCT AAGTCACCTA 1563
2) INFORMATION FOR SEQ ID N0:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 200 amino acids
(B) TYPE: Amino Acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Protein
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Sus scrofa
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:4:
Met Leu Gly Lys Cys Leu Thr Ala Gly Cys Cys Ser Gln Leu Pro Phe
1 5 10 15
Leu Trp Cys Ile Val Pro Phe Cys Phe Ala Ala Leu Val Asn Ala Ser
20 25 30
Ser Ser Ser Ser Ser Gln Leu Gln Ser Ile Tyr Asn Leu Thr Ile Cys
35 40 45
Glu Leu Asn Gly Thr Asp Trp Leu Asn Lys Asn Phe Asp Trp Ala Val
50 55 60
Glu Thr Phe Val Ile Phe Pro Val Leu Thr His Ile Val Ser Tyr Gly
65 70 75 80
Ala Leu Thr Thr Ser His Phe Leu Asp Ala Val Gly Leu Ile Thr Val
85 90 95
Ser Thr Ala Gly Tyr Tyr His Gly Arg Tyr Val Leu Ser Ser Val Tyr
100 105 110
Ala Val Cys Ala Leu Ala Ala Leu Ile Cys Phe Val Ile Arg Leu Thr
115 120 125
Lys Asn Cys Met Ser Trp Arg Tyr Ser Cys Thr Arg Tyr Thr Asn Phe
130 135 140
Leu Leu Asp Ser Lys Gly Lys Leu Tyr Arg Trp Arg Ser Pro Val Ile
145 150 155 160
Ile Glu Lys Gly Gly Lys Val Glu Val Asp Gly His Leu Ile Asp Leu
165 170 175
Lys Arg Val Val Leu Asp Gly Ser Ala Ala Thr Pro Val Thr Lys Val
180 185 190
Ser Ala Glu Gln Trp Cys Arg Pro
195 200
(2) INFORMATION FOR SEQ ID N0:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 38 base pairs
(B) TYPE: Nucleic acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
CA 02418780 2003-02-28
(ii) MOLECULE TYPE: DNA
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Artificial Sequence
(ix) FEATURE:
(A) NAME/KEY:
(B) LOCATION:
(D) OTHER INFORMATION: wtORF5 forward primer
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:5:
CCGGATCCGC CGCCGCCATG TTGGGGAAAT GCCTGACC 3B
(2) INFORMATION FOR SEQ ID N0:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 39 base pairs
(B) TYPE: Nucleic acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(ii) MOLECULE TYPE: DNA
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Artificial Sequence
(ix) FEATURE:
(A) NAME/KEY:
(B) LOCATION:
(D) OTHER INFORMATION: synORFS forward primer
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:6:
CATGGATCCG CCGCCGCCAT GCTGGGCAAG TGCTTGACC 39
(2) INFORMATION FOR SEQ ID N0:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs
(B) TYPE: Nucleic acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(ii) MOLECULE TYPE: DNA
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Artificial Sequence
(ix) FEATURE:
(A) NAME/KEY:
(B) LOCATION:
(D) OTHER INFORMATION: ORF5 reverse primer
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:7:
TCTAGAGGCA AAAGTCATCT AGGG 24
6
CA 02418780 2003-02-28
(2) INFORMATION FOR SEQ ID N0:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 620 base pairs
(B) TYPE: Nucleic acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(ii) MOLECULE TYPE: DNA
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Artificial Sequence
(ix) FEATURE:
{A) NAME/KEY:CDS
(B) LOCATION: (10)...(612)
(D) OTHER INFORMATION: synORFS encoding GP5 of PRRSV strain IAF-94-
287 (Figure 1)
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:8:
ACCGGATCC ATG CTG GGC AAG TGC CTG ACC GCC GGC TGT TGC TTC CAG CTG 51
Met Leu Gly Lys Cys Leu Thr Ala Gly Cys Cys Phe Gln Leu
1 5 10
CCC TTC CTG TGG TGT ATG GTG CCG TTC TGT TTG GCC GCC CTG GTG AAC 99
Pro Phe Leu Trp Cys Met Val Pro Phe Cys Leu Ala Ala Leu Val Asn
15 20 25 30
GCC TCC TCC TCC TCC TCC TCC CAG CTG CAG TCC ATG TAG AAC CTG ACG 147
Ala Ser Ser Ser Ser Ser Ser Gln Leu Gln Ser Met Asn Leu Thr
35 40 45
ATG TGT GAG CTG AAC GGC ACG GAC TGG CTG AAC AAG AAC TTG GAC TGG 195
Met Cys Glu Leu Asn Gly Thr Asp Trp Leu Asn Lys Asn Leu Asp Trp
50 55 60
GCC GTG GAG ACC TTC GTG ATC TTC CCC GTG CTG ACC CAC ATC GTG TCC 243
Ala Val Glu Thr Phe Val Ile Phe Pro Val Leu Thr His Ile Val Ser
65 70 75
TAC GGC GCC CTG ACC ACC TCC CAC TTC CTG GAC GCC GTG GGC CTG ATC 291
Tyr Gly Ala Leu Thr Thr Ser His Phe Leu Asp Ala Val Gly Leu Ile
80 85 90
ACC GTG TCC ACC GCC GGC TAC TAC CAC GGC CGC TAC GTG CTG TCC TCC 339
Thr Val Ser Thr Ala Gly Tyr Tyr His Gly Arg Tyr Val Leu Ser Ser
95 100 105
GTG TAC GCC GTG TGC GCC CTG GCC GCC CTG ATC TGC TTC GTG ATC CGC 387
Val Tyr Ala Val Cys Ala Leu Ala Ala Leu Ile Cys Phe Val Ile Arg
110 115 120 125
CTG ACC AAG AAC TGC ATG TCC TGG CGC TAC TCC TGT ACC CGC TAC ACC 435
Leu Thr Lys Asn Cys Met Ser Trp Arg Tyr Ser Cys Thr Arg Tyr Thr
130 135 140
AAC TTC CTG CTG GAC TCC AAG GGC AAG CTG TAC CGC TGG CGC TCC CCC 483
Asn Phe Leu Leu Asp Ser Lys Gly Lys Leu Tyr Arg Trp Arg Ser Pro
CA 02418780 2003-02-28
145 150 155
GTG ATC ATC GAG AAG GGC GGC AAG GTG GAG GTG GAC GGC CAC CTG ATC 531
Val Ile Ile Glu Lys Gly Gly Lys Val Glu Val Asp Gly His Leu Ile
160 165 170
GAC CTG AAG CGC GTG GTG CTG GAC GGC TCC GCC GCC ACC CCC GTG ACC 579
Asp Leu Lys Arg Val Val Leu Asp Gly Ser Ala Ala Thr Pro Val Thr
175 180 185
AAG GTG TCC GCC GAG CAG TGG TGT CAC CCC TAG GATCCGCC 620
Lys Val Ser Ala Glu Gln Trp Cys His Pro
190 195
2) INFORMATION FOR SEQ ID N0:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH:199 amino acids
(B) TYPE: Amino Acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Protein
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Artificial Sequence
(ix) FEATURE:
(A) NAME/KEY:
(B) LOCATION:
(D) OTHER INFORMATION: synORFS of PRRSV strain IAF-KLOP
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:9:
Met Leu Gly Lys Cys Leu Thr Ala Gly Cys Cys Phe Gln Leu Pro Phe
1 5 10 15
Leu Trp Cys Met Val Pro Phe Cys Leu Ala Ala Leu Val Asn Ala Ser
20 25 30
Ser Ser Ser Ser Ser Gln Leu Gln Ser Met Asn Leu Thr Met Cys Glu
35 40 45
Leu Asn Gly Thr Asp Trp Leu Asn Lys Asn Leu Asp Trp Ala Val Glu
50 55 60
Thr Phe Val Ile Phe Pro Val Leu Thr His Ile Val Ser Tyr Gly Ala
65 70 75 80
Leu Thr Thr Ser His Phe Leu Asp Ala Val Gly Leu Ile Thr Val Ser
85 90 95
Thr Ala Gly Tyr Tyr His Gly Arg Tyr Val Leu Ser Ser Val Tyr Ala
100 105 110
Val Cys Ala Leu Ala Ala Leu Ile Cys Phe Val Ile Arg Leu Thr Lys
115 120 125
Asn Cys Met Ser Trp Arg Tyr Ser Cys Thr Arg Tyr Thr Asn Phe Leu
130 135 140
Leu Asp Ser Lys Gly Lys Leu Tyr Arg Trp Arg Ser Pro Val Ile Ile
145 150 155 160
Glu Lys Gly Gly Lys Val Glu Val Asp Gly His Leu Ile Asp Leu Lys
165 170 175
Arg Val Val Leu Asp Gly Ser Ala Ala Thr Pro Val Thr Lys Val Ser
180 185 190
Ala Glu Gln Trp Cys His Pro
195
8
CA 02418780 2003-02-28
(2) INFORMATION FOR SEQ ID NO:10:
(i) SEQUENCE
CHARACTERISTICS:
(A) LENGTH: basepairs
1563
(B) TYPE:Nucleic acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(ii)MOLECULE DNA
TYPE:
(vi)ORIGINAL
SOURCE:
(A) ORGANISM:Sus scrofa
(ix)FEATURE:
(A) NAME/KEY:CDS
(B) LOCATION: (1)...(603)
(D) OTHERINFORMATION : ORF encoding GP5 of PRRSV
wt strain
IAF-BAJ Accession
GenBank #
U64929
(xi)SEQUENCE SEQID
DESCRIPTION: NO;10:
ATGTTG GGG AAA TTG ACCGCGGGCTGT TGCTCGCAA TTGCCTTTT 48
TGC
MetLeu Gly Lys Leu ThrAlaGlyCys CysSerGln LeuProPhe
Cys
1 5 10 15
TTGTGG TGT ATC CCG TTCTGTTTTGCT GCGCTCGTC AACGCCAGC 96
GTG
LeuTrp Cys Ile Pro PheCysPheAla AlaLeuVal AsnAlaSer
Val
20 25 30
AACAAC AGC AGC CAA TTGCAGTCGATT TATAACCTG ACGATATGC 144
TCC
AsnAsn Ser Ser Gln LeuGlnSerIle TyrAsnLeu ThrIleCys
Ser
35 40 45
GAGCTG AAT GGC GAT TGGCTGAATAAA AATTTTGAT TGGGCAGTG 192
ACA
GluLeu Asn Gly Asp TrpLeuAsnLys AsnPheAsp TrpAlaVal
Thr
50 55 60
GAGACT TTT GTT TTT CCTGTGCTGACT CACATTGTC TCCTATGGC 240
ATC
GluThr Phe Val Phe ProValLeuThr HisIleVal SerTyrGly
Ile
65 70 75 80
GCCCTC ACC ACC CAT TTCCTTGACACA GTCGGTCTG ATCACTGTG 288
AGC
AlaLeu Thr Thr His PheLeuAspThr ValGlyLeu IleThrVal
Ser
85 90 95
TCTACC GCC GGA TAC CACGGGCGGTAT GTCTTGAGC AGCGTCTAC 336
TAT
SerThr Ala Gly Tyr HisGlyArgTyr ValLeuSer SerValTyr
Tyr
100 105 110
GCTGTC TGC GCC GCT GCGCTAATTTGC TTCGTCATT AGGTTGACG 384
TTG
AlaVal Cys Ala Ala AlaLeuIleCys PheValIle ArgLeuThr
Leu
115 120 125
AAAAAC TGC ATG TGG CGCTACTCATGT ACCAGATAT ACCAACTTT 432
TCC
LysAsn Cys Met Trp ArgTyrSerCys ThrArgTyr ThrAsnPhe
Ser
130 135 140
CTTCTG GAC TCC GGC AAACTCTATCGT TGGCGGTCA CCCGTCATC 480
AAG
LeuLeu Asp Ser Gly LysLeuTyrArg TrpArgSer ProValIle
Lys
9
CA 02418780 2003-02-28
145 150 155 160
ATA GAG AAA GGG GGT AAA GTT GAG GTT GAT GGT CAT CTG ATC GAC CTC 528
Ile Glu Lys Gly Gly Lys Val Glu Val Asp Gly His Leu Ile Asp Leu
165 170 175
AAG AGA GTT GTG CTT GAT GGT TCC GCG GCA ACC CCT GTA ACC AAA GTT 576
Lys Arg Val Val Leu Asp Gly Ser Ala Ala Thr Pro Val Thr Lys Val
180 185 190
TCA GCG GAA CAA TGG TGT CGT CCC TAG ACGACTTCTG CAATGACAGC 623
Ser Ala Glu Gln Trp Cys Arg Pro
195 200
ACGGCTCCGC AAAAGGTGCT CCTGGCGTTT TCTATCACCT ACACGCCAGT AATGATATAT 683
GCCCTAAAGG TAAGTCGCGG CCGACTGCTA GGGCTTCTGC ACCTTTTAAT TTTCCTGAAT 743
TGTGCTTTTA CCTTCGGGTA TATGACATTC GCGCACTTTC AGAGTACAAA TAAAGTCGCG 803
CTCACTATGG GAGCAGTAGT TGCGCTCCTT TGGGGGGTGT ACTCAGCCAT AGAAACCTGG 863
AAATTCATCA CCTCCAGATG CCGTTTGTGC TTGCTAGGCC GCAAGTACAT TCTGGCCCCT 923
GCCCACCACG TTGAGAGTGC CGCAGGCTTT CATCCGATTG CGGCAAGTGA TAACCACGCA 983
TTTGTCGTCC GGCGTCCCGG CTCCACTACG GTTAACGGCA CATTGGTGCC CGGGTTGAAA 1043
AGCCTCGTGT TGGGTGGCAG AAAAGCTGTC AAACGGGGAG TGGTAAACCT CGTTAAATAT 1103
GCCAAATAAC AACGGCAAGC AGCAGAAGAA AAAGAAAGGG GATGGCCAGC CAGTCAATCA 1163
GCTGTGCCAG ATGCTGGGTA AGATCATCGC CCAGCAAAAC CAGTCCAGAG GCAAGGGACC 1223
GGGGAAGAAA AATAAGAAGA AAAACCCGGA GAAGCCCCAT TTTCCTCTAG CGACTGAAGA 1283
TGACGTCAGA CATCACTTCA CCCCTAGTGA GCGGCAATTG TGTCTGTCGT CAATCCAGAC 1343
TGCCTTTAAT CAAGGCGCTG GAACTTGCAC CCTATCAGAT TCAGGGAGAA TAAGTTACGC 1403
TGTGGAGTTT AGTTTGCCTA CGCATCATAC TGTGCGCCTG ATTCGCGTCA CAGCATCACC 1463
CTCAGCATGA TGAGCTGGCA TTCTTGAGAC ATCCCAGTGT TTGAATTGGA AGAATGTGTG 1523
GTGAATGGCA CTGATTGATA TTGTGCCTCT AAGTCACCTA 1563
2) INFORMATION FOR SEQ ID NO:11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH:200 amino acids
(B) TYPE: Amino Acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Protein
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Sus scrofa
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:11:
Met Leu Gly Lys Cys Leu Thr Ala Gly Cys Cys Ser Gln Leu Pro Phe
1 5 10 15
Leu Trp Cys Ile Val Pro Phe Cys Phe Ala Ala Leu Val Asn Ala Ser
20 25 30
Asn Asn Ser Ser Ser Gln Leu Gln Ser Ile Tyr Asn Leu Thr Ile Cys
35 40 45
Glu Leu Asn Gly Thr Asp Trp Leu Asn Lys Asn Phe Asp Trp Ala Val
50 55 60
Glu Thr Phe Val Ile Phe Pro Val Leu Thr His Ile Val Ser Tyr Gly
65 70 75 80
Ala Leu Thr Thr Ser His Phe Leu Asp Thr Val Gly Leu Ile Thr Val
85 90 95
Ser Thr Ala Gly Tyr Tyr His Gly Arg Tyr Val Leu Ser Ser Val Tyr
100 105 110
Ala Val Cys Ala Leu Ala Ala Leu Ile Cys Phe Val Ile Arg Leu Thr
1~
CA 02418780 2003-02-28
115 120 125
Lys Asn Cys Met Ser Trp Arg Tyr Ser Cys Thr Arg Tyr Thr Asn Phe
130 135 140
Leu Leu Asp Ser Lys Gly Lys Leu Tyr Arg Trp Arg Ser Pro Val Ile
145 150 155 160
Ile Glu Lys Gly Gly Lys Val Glu Val Asp Gly His Leu Ile Asp Leu
165 170 175
Lys Arg Val Val Leu Asp Gly Ser Ala Ala Thr Pro Val Thr Lys Val
180 185 190
Ser Ala Glu Gln Trp Cys Arg Pro
195 200
(2) INFORMATION FOR SEQ ID N0:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1563base rs
pai
(B) TYPE: Nucleicacid
(C) STRANDEDNESS:Single
(D) TOPOLOGY: ear
Lin
(ii)MOLECULE TYPE:
DNA
(vi)ORIGINAL SOURCE:
(A) ORGANISM: scrofa
Sus
(ix)FEATURE:
(A) NAME/KEY:CDS
(B) LOCATION: ...(603)
(1)
(D) OTHER INFORMATION: encoding PRRSV
wtORF5 GP5 strain
of
IAF-DESR GenBank cession 930
Ac #U64
(xi)SEQUENCE DESCRIPTION: ID
SEQ N0:12:
ATGTTG GGG AAA TGC TTG GTG TAT TGCTCGCAA TTGCCTTTT 48
ACC GGC
MetLeu Gly Lys Cys Leu Val Tyr CysSerGln LeuProPhe
Thr Gly
1 5 10 15
TTGTGG TGT ATC GTG CCG TGT GCT GCGCTCGTC AACGCCAGC 96
TTC TTT
LeuTrp Cys Ile Val Pro Cys Ala AlaLeuVal AsnAlaSer
Phe Phe
20 25 30
AGCACC AGC AGC TCC CAC CAG ATT TATAACCTG ACAATATGC 144
TTA TTG
SerThr Ser Ser Ser His Gln Ile TyrAsnLeu ThrIleCys
Leu Leu
35 40 45
GAGCTG AAT GGC ACA GAT CTG GAA AAATTTGAT TGGGCAGTG 192
TGG AAT
GluLeu Asn Gly Thr Asp Leu Glu LysPheAsp TrpAlaVal
Trp Asn
50 55 60
GAGACT TTT GTT ATC TTT GTG ACT CATATTGTC TCCTATGGC 240
CCT TTG
GluThr Phe Val Ile Phe Val Thr HisIleVal SerTyrGly
Pro Leu
65 70 75 80
GCCCTC ACT ACC AGC CAT CTT ACA GTCGGTCTT GTCACTGTG 288
TTC GAC
AlaLeu Thr Thr Ser His Leu Thr ValGlyLeu ValThrVal
Phe Asp
85 90 95
TCTACC GCC GGA TAT TAC GGG TAT GTCTTGAGT AGCATCTAC 336
CAT CGG
SerThr Ala Gly Tyr Tyr Gly Tyr ValLeuSer SerIleTyr
His Arg
100 105 110
11
CA 02418780 2003-02-28
GCT GTC TGT GCC CTG GCT GCG TTG ATT TGC TTC GTC ATT AGG TTG GTG 384
Ala Val Cys Ala Leu Ala Ala Leu Ile Cys Phe Val Ile Arg Leu Val
115 120 125
AAG AAC TGC ATG TCC TGG CGC TAC TCA TGT ACC AGA TAT ACT AAC TTT 432
Lys Asn Cys Met Ser Trp Arg Tyr Ser Cys Thr Arg Tyr Thr Asn Phe
130 135 140
CTT CTG GAC ACC AAG GGC AAA CTC TAT CGC TGG CGG TCG CCC GTC ATC 480
Leu Leu Asp Thr Lys Gly Lys Leu Tyr Arg Trp Arg Ser Pro Val Ile
145 150 155 160
GTA GAG AAA AGG GGT AAA GTT GAG GTT GGA GGT CAC CTT ATC GAC CTC 528
Val Glu Lys Arg Gly Lys Val Glu Val Gly Gly His Leu Ile Asp Leu
165 170 175
AAG AGA GTT GTG CTT GAC GGT TCC GCG GCA ACC CCT GTA ACC AGA GTT 576
Lys Arg Val Val Leu Asp Gly Ser Ala Ala Thr Pro Val Thr Arg Val
180 185 190
TCA GCG GAA CAA TGG GGT CGT CCC TAG ATGACTTTTG CCATGACAGC 623
Ser Ala Glu Gln Trp Gly Arg Pro
195 200
ACAGCTCCAC AAAAGGTGCT CTTGGCGTTT TCTATCACCT ACACGCCAGT ATTGATATAT 683
GCCCTGAAGG TGAGTCGCGG CCGACTACTA GGGCTTCTGC ACCTTTTAAT TTTTCTGAAT 743
TGTGCTTTCA CCTTCGGGTA TATGACATTC ACGCACTTTC AGAGCACAAA TAAGGTCGCG 803
CTCACTATGG GAGCAGTGGT TGCACTCCTT TGGGGGGTGT ACTCAGCCAT AGAAACCTGG 863
AAATTCATCA CTTCCAGATG CCGTTTGTGC TTGCTAGGCC GCAAGTACAT TCTGGCCCCT 923
GCCCACCACG TTGAGAGTGC CGCAGGCTTT CATCCGATTG CGGCAAGTGA TAACCACGCA 983
TTTGTCGTCC GGCGTCCCGG CTCTACTACG GTCAACGGCA CATTGGTGCC CGGGTTGAAA 1043
AGCCTCGTGT TGGGTGGCAA AAAGGCTGTC AAGCGGGGAG TGGTAAACCT CGTTAAATAT 1103
GCCAAATAAC AACGGCAGGC AGCAGAAGAA GAAGAAGGGG GATGGCCAGC CAGTCAATCA 1163
GCTGTGCCAG ATGCTGGGTA AGATCATCGC CCAGCAAAAC CAGTCCAGAG GTAAGGGACC 1223
GGGGAAGAAA AACAAGAAGA AAAACCCGGA AAAGCCCCAT TTTCCTCTAG CGACTGAAGA 1283
TGACGTCAGA CATCACTTCA CCCCTAGTGA GCGGCAATTG TGTTTGTCGT CAATCCAGAC 1343
TGCCTTCAAT CAAGGCGCTG GAACTTGCAC CCTGTCGGAT TCAGGGAGGA TAAGTTACGC 1403
TGTGGAGTTC AGTTTGCCTA CGCATCATAC TGTACGCTTA ATTCGCGTCA CAGCATCACC 1463
CTCAGCATGA TGAGCTGGCA TTCTTGAGAC ATCCCAGTGT TTGAATTGGA AGAATGTGTG 1523
GTGAATGGCA CTGATTGATA TTGTGCCTCT AAGTCACCTA 1563
2) INFORMATION FOR SEQ ID N0:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH:200 amino acids
(B) TYPE: Amino Acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Protein
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Sus scrofa
(xi) SEQUENCE DESCRIPTION: 5EQ ID N0:13:
Met Leu GIy Lys Cys Leu Thr Val Gly Tyr Cys Ser GIn Leu Pro Phe
1 5 10 15
Leu Trp Cys Ile Val Pro Phe Cys Phe Ala Ala Leu Val Asn Ala Ser
20 25 30
12
CA 02418780 2003-02-28
Ser Thr Ser Ser Ser His Leu Gln Leu Ile Tyr Asn Leu Thr Ile Cys
35 40 45
Glu Leu Asn Gly Thr Asp Trp Leu Asn Glu Lys Phe Asp Trp Ala Val
50 55 60
Glu Thr Phe Val Ile Phe Pro Val Leu Thr His Ile Val Ser Tyr Gly
65 70 75 80
Ala Leu Thr Thr Ser His Phe Leu Asp Thr Val Gly Leu Val Thr Val
85 90 95
Ser Thr Ala Gly Tyr Tyr His Gly Arg Tyr Val Leu Ser Ser Ile Tyr
100 105 110
Ala Val Cys Ala Leu Ala Ala Leu Ile Cys Phe Val Ile Arg Leu Val
115 120 125
Lys Asn Cys Met Ser Trp Arg Tyr Ser Cys Thr Arg Tyr Thr Asn Phe
130 135 140
Leu Leu Asp Thr Lys Gly Lys Leu Tyr Arg Trp Arg Ser Pro Val Ile
145 150 155 160
Val Glu Lys Arg Gly Lys Val Glu Val Gly Gly His Leu Ile Asp Leu
165 170 175
Lys Arg Val Val Leu Asp Gly Ser Ala Ala Thr Pro Val Thr Arg Val
180 185 190
Ser Ala Glu Gln Trp Gly Arg Pro
195 200
(2) INFORMATION FOR SEQ ID N0:14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1566 base pairs
(B) TYPE: Nucleic acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(ii) MOLECULE TYPE: DNA
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Sus scrofa
(ix) FEATURE:
(A) NAME/KEY:CDS
(B) LOCATION: (1) ... (603)
(D) OTHER INFORMATION: wtORF5 encoding GP5 of PRRSV strain
IAF-653 GenBank Accession #U64931
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:14:
ATG TTG GGG AAA TGC TTG ACC GCG GGT TGT TGC TCG CAA TTG CCT TTT 48
Met Leu Gly Lys Cys Leu Thr Ala Gly Cys Cys Ser Gln Leu Pro Phe
1 5 10 15
TTG TGG TGT ATC GTG CCG TTC TGT TTT GTT GCG CTC GTC AAC GCC AAC 96
Leu Trp Cys Ile Val Pro Phe Cys Phe Val Ala Leu Val Asn Ala Asn
20 25 30
ACA GAC AGC AGC TCC CAT TTA CAG TTG ATT TAT AAC CTG ACG ATA TGC 144
Thr Asp Ser Ser Ser His Leu Gln Leu Ile Tyr Asn Leu Thr Ile Cys
35 40 45
GAG CTG AAT GGC ACA GAT TGG CTG AAT GAC AAA TTT GAT TGG GCA GTG 192
Glu Leu Asn Gly Thr Asp Trp Leu Asn Asp Lys Phe Asp Trp Ala Val
50 55 60
13
CA 02418780 2003-02-28
GAG ACT TTT GTC ATC TTT CCT GTG TTG ACT CAT ATT GTC TCC TAT GGC 240
Glu Thr Phe Val Ile Phe Pro Val Leu Thr His Ile Val Ser Tyr Gly
65 70 75 80
GCC CTC ACC ACC AGC CAT TTC CTT GAC ACA GTC GGT CTG ATC ACT GTG 288
Ala Leu Thr Thr Ser His Phe Leu Asp Thr Val Gly Leu Ile Thr Val
85 90 95
TCT ACC GCC GGA TAT TAC CAC GGG CGG TAT GTC TTG AGT AGC ATC TAT 336
Ser Thr Ala Gly Tyr Tyr His Gly Arg Tyr Val Leu Ser Ser Ile Tyr
100 105 110
GCT GTC TGT GCC CTG GCA GCG TTG ATT TGC TTC GCC ATT AGG TTG ACG 384
Ala Val Cys Ala Leu Ala Ala Leu Ile Cys Phe Ala Ile Arg Leu Thr
115 120 125
AAG AAC TGC ATG TCC TGG CGC TAC TCA TGC ACC AGA TAT ACC AAC TTT 432
Lys Asn Cys Met Ser Trp Arg Tyr Ser Cys Thr Arg Tyr Thr Asn Phe
130 135 140
CTT CTG GAC ACT AAG GGC AAA CTC TAT CGT TGG CGG TCA CCC GTC ATC 480
Leu Leu Asp Thr Lys Gly Lys Leu Tyr Arg Trp Arg Ser Pro Val Ile
145 150 155 160
ATA GAA AGA CAG GGT AAA GTT GAG GTT GAA GGT CAC CTG ATC GAC CTC 528
Ile Glu Arg Gln Gly Lys Val Glu Val Glu Gly His Leu Ile Asp Leu
165 170 175
AAG AGA GTT GTG CTT GAC GGT TCC GCG GCA ACC CCT GTA ACC AGA GTT 576
Lys Arg Val Val Leu Asp Gly Ser Ala Ala Thr Pro Val Thr Arg Val
180 185 190
TCA GCG GAA CGA TGG GGT CGT CCC TAG ACGACTTTTG CCATGACAGC 623
Ser Ala Glu Arg Trp Gly Arg Pro
195 200
ACGGCTCCAC AAAAGGTGCT TTTGGCGTTT TCTATTACTT ACACGCCAGT AATGATATAT 683
GCTCTAAAGG TAAGTCGTGG GCGACTGCTA GGGCTTCTGC ACCTTTTAAT TTTCCTGAAT 743
TGTGCTTTCA CCTTCGGGTA TATGACATTC ACGCACTTTC GGAGCACAAA TAAGGTCGCG 803
CTCACTATGG GAGCGGTGAT CGCACTCCTT TGGGGGGTGT ATTCAGCCAT AGAGACCTGG 863
AGATTCATCA CCTCCAGATG CCGTTTGTGC TTGTTAGGCC GCAAGTACAT TCTGGCCCCT 923
GCCCACCACG TTGAAAGTGC CGCAGGCTTT CATCCGATTG CGACGAGTGA TAACCACGCA 983
TTTGTCGTTC GGCGTCCCGG CTCCACTACG GTTAACGGCA CATTGGTGCC CGGGTTGAAA 1043
AGCCTCGTGT TGGGTGGCAA AAAAGCTGTC AAACAGGGAG TGGTAAACCT TGTTAAATAT 1103
GCCAAATAAT AACGGCAAGC AGCAAAAGAA AAAGAAGGGG GATGGCCAGC CAGTCAATCA 1163
GCTGTGTCAG ATGCTGGGTA AGATCATCGC CCAGCAAAAC CAGTCCAGAG GTAGGGGACC 1223
GGGAAACAAG AAAAATAAGA AGAAAAACCC GGAAAAGCCC CATTTTCCTC TAGCGACTGA 1283
AGATGACGTC AGACATCACT TCACCCCTAG TGAGCGGCAA CTGTGTCTGT CGTCAATCCA 1343
GACCGCCTTT AATCAAGGCG CTGGAACTTG CACCCTGTCG GATTCAGGGA GGATAAGTTA 1403
CGCTGTGGAG TTCAGTTTGC CTACGCATCA TACTGTGCGC CTGATTCGCG TTACAGCATC 1463
ACCCTCAGCA TGATGAGCTG GCATTCTTGA GACATCCCAG TGTTCAAATT GGAAGAATGT 1523
GTGGTGAATG GCACTGATTG ATATTGTGCC TCTAAGTCAC CTA 1566
2) INFORMATION FOR SEQ ID N0:15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH:200 amino acids
(B) TYPE: Amino Acid
(D) TOPOLOGY: linear
14
CA 02418780 2003-02-28
(ii) MOLECULE TYPE: Protein
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Sus scrofa
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:15:
Met Leu Gly Lys Cys Leu Thr Ala Gly Cys Cys Ser Gln Leu Pro Phe
1 5 10 15
Leu Trp Cys Ile Val Pro Phe Cys Phe Val Ala Leu Val Asn Ala Asn
20 25 30
Thr Asp Ser Ser Ser His Leu Gln Leu Ile Tyr Asn Leu Thr Ile Cys
35 40 45
Glu Leu Asn Gly Thr Asp Trp Leu Asn Asp Lys Phe Asp Trp Ala Val
50 55 60
Glu Thr Phe Val Ile Phe Pro Val Leu Thr His Ile Val Ser Tyr Gly
65 70 75 80
Ala Leu Thr Thr Ser His Phe Leu Asp Thr Val Gly Leu Ile Thr Val
85 90 95
Ser Thr Ala GIy Tyr Tyr His Gly Arg Tyr Val Leu Ser Ser Ile Tyr
100 105 110
Ala Val Cys Ala Leu Ala Ala Leu Ile Cys Phe Ala Ile Arg Leu Thr
115 120 125
Lys Asn Cys Met Ser Trp Arg Tyr Ser Cys Thr Arg Tyr Thr Asn Phe
130 135 140
Leu Leu Asp Thr Lys Gly Lys Leu Tyr Arg Trp Arg Ser Pro Val Ile
145 150 155 160
Ile Glu Arg Gln Gly Lys Val Glu Val Glu Gly His Leu Ile Asp Leu
165 170 175
Lys Arg Val Val Leu Asp Gly Ser Ala Ala Thr Pro Val Thr Arg Val
180 185 190
Ser Ala Glu Arg Trp Gly Arg Pro
195 200
(2) INFORMATION FOR SEQ ID N0:16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1563 base pairs
(B) TYPE: Nucleic acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(ii) MOLECULE TYPE: DNA
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Sus scrofa
(ix) FEATURE:
(A) NAME/KEY:CDS
(B) LOCATION: (1)...(603)
(D) OTHER INFORMATION: wtORFS encoding GP5 of PRRSV strain
IAF-93-2616 GenBank Accession #U64932
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:16:
ATG TTG GGG AAA TGC TTG ACC GCG GGC TGT TGC TCG CGA TTG CCT TTT 48
Met Leu Gly Lys Cys Leu Thr Ala Gly Cys Cys Ser Arg Leu Pro Phe
IS
CA 02418780 2003-02-28
1 5 10 15
TTG TGG TGT ATC GTG CCG TTC TGT TTT GCT GCG CTC GTC AAC GCC AGC 96
Leu Trp Cys Ile Val Pro Phe Cys Phe Ala Ala Leu Val Asn Ala Ser
20 25 30
AAC AGC AGC AGC TCC CAT TTA CAG TTG ATT TAT AAC CTG ACG ATA TGT 144
Asn Ser Ser Ser Ser His Leu Gln Leu Ile Tyr Asn Leu Thr Ile Cys
35 40 45
GAG CTG AAT GGC ACA GAT TGG CTG GAC AAA AAA TTT GAT TGG GCA GTT 192
Glu Leu Asn Gly Thr Asp Trp Leu Asp Lys Lys Phe Asp Trp Ala Val
50 55 60
GAG ACT TTC GTT ATT TTT CCT GTG TTG ACT CAC ATT GTC TCC TAT GGC 240
Glu Thr Phe Val Ile Phe Pro Val Leu Thr His Ile Val Ser Tyr Gly
65 70 75 80
GCC CTT ACC ACC AGC CAT TTC CTT GAC ACA GTC GGT CTG ATC ACT GTG 288
Ala Leu Thr Thr Ser His Phe Leu Asp Thr Val Gly Leu Ile Thr Val
85 90 95
TCG ACC GCC GGA TAT TAC CAC GGG CGG TAT GTC TTG AGT AGC ATC TAC 336
Ser Thr Ala Gly Tyr Tyr His Gly Arg Tyr Val Leu Ser Ser Ile Tyr
100 105 110
GCT GTC TGT GCC CTG GCC GCG TTG ATT TGC TTC GTC ATC AGG TTG ACG 384
Ala Val Cys Ala Leu Ala Ala Leu Ile Cys Phe Val Tle Arg Leu Thr
115 120 125
AAG AAC TGC ATG TCC TGG CGC TAC TCA TGC ACT AGA TAT ACC AAC TTT 432
Lys Asn Cys Met Ser Trp Arg Tyr Ser Cys Thr Arg Tyr Thr Asn Phe
130 135 140
CTT CTG GAC ACC AAG GGC AAA CTC TAT CGT TGG CGG TCG CCC GTC ATT 480
Leu Leu Asp Thr Lys Gly Lys Leu Tyr Arg Trp Arg Ser Pro Val Ile
145 150 155 160
ATA GAG AAA AGG GGT AAG GTT GAG GTT GAG GGT CAA CTT ATC GAC CTC 528
Ile Glu Lys Arg Gly Lys Val Glu VaI Glu Gly Gln Leu Ile Asp Leu
165 170 175
AAG AGA GTC GTG CTT GAT GGT TCC GCG GCA ACC CCT ATA ACC AAA GTT 576
Lys Arg Val Val Leu Asp Gly Ser Ala Ala Thr Pro Ile Thr Lys Val
180 185 190
TCA GCG GAA CAA TGG GGT CGT CCC TAG ATGACTTTTG CCATGACAGC 623
Ser Ala Glu Gln Trp Gly Arg Pro
195 200
TCGGCTCCAC AAAAGGTGCT CTTGGCGTTT TCCATTACCT ACACGCCAGT GATGATATAT 683
GCCCTGAAGG TAAGTCGCGG CCGACTGCTA GGGCTTCTGC ACCTTTTGAT TTTCCTGAAT 743
TGTGCTTTCA CCTTCGGGTA CATGACATTC ACGCACTTTC AGAGTACAAA TAGGGTCGCG 803
CTCACTATGG GAGCSGTAAT TGCACTCCTC TGGGGAGTGT ACTCAGCCAT AGAGACCTGG 863
AGATTCATCA CCTCCAGATG CCGCTTGTGC TTGTTAGGTC GCAAGTACAT TCTGGCCCCT 923
GCCCACCACG TTGAAAGTGC CGCAGGTTTT CATCCGATTG CGACAAGTGA TAACCACGCA 983
TTTGTCGTCC GGCGTCCCGG CTCCACTACG GTTAACGGCA CATTGGTGCC CGGGTTGAAA 1043
AGTCTCGTGT TGGGTGGCAG AAAAGCTGTC AAACAGGGAG TGGTAAACCT TGTCAAATAT 1103
GCCAAATAAC AACGGCAGGC AGCAAAAGAA AAAGAAGGGG GATGGCCAGC CAGTCAACCA 1163
GCTGTGCCAA ATGCTGGGTA AGATCATCGC TCAGCAAAAC CAGTCCAGAG GTAAGGGACC 1223
GGGGAAGAAA AATAAGAAGA AAAACCCGGA AAAGCCCCAT TTTCCTCTAG CGACTGAAGA 1283
16
CA 02418780 2003-02-28
TGACGTCAGA CATCACTTTA CCCCTAGTGA GCGGCAATTG TGTCTGTCGT CAATCCAGAC 1343
TGCCTTCAAT CAAGGCGCTG GAACTTGCAC CCTCTCAGAT TCAGGGAGGA TAAGTTACAC 1403
TGTGGAGTTT AGTTTGCCTA CGCATCATAC TGTGCGCCTG ATTCGCGTTA CAGCATCACC 1463
CTCAGCATGA TGAGCTGGCA TTCTTGAGAC ATCCCAGTGT TTGAATTGGA AGAATGTGTG 1523
GTGAATGGCA CTGATTGATA TTGTGCCTCT AAGTCACCTA 1563
2) INFORMATION FOR SEQ ID N0:17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH:200 amino acids
(B) TYPE: Amino Acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Protein
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Sus scrofa
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:17:
Met Leu Gly Lys Cys Leu Thr Ala Gly Cys Cys Ser Arg Leu Pro Phe
1 5 10 15
Leu Trp Cys Ile Val Pro Phe Cys Phe Ala Ala Leu Val Asn Ala Ser
20 25 30
Asn Ser Ser Ser Ser His Leu Gln Leu Ile Tyr Asn Leu Thr Ile Cys
35 40 45
Glu Leu Asn Gly Thr Asp Trp Leu Asp Lys Lys Phe Asp Trp Ala Val
50 55 60
Glu Thr Phe Val Ile Phe Pro Val Leu Thr His Ile Val Ser Tyr Gly
65 70 75 80
Ala Leu Thr Thr Ser His Phe Leu Asp Thr Val Gly Leu Ile Thr Val
85 90 95
Ser Thr Ala Gly Tyr Tyr His Gly Arg Tyr Val Leu Ser Ser Ile Tyr
100 105 110
Ala Val Cys Ala Leu Ala Ala Leu Ile Cys Phe Val Ile Arg Leu Thr
115 120 125
Lys Asn Cys Met Ser Trp Arg Tyr Ser Cys Thr Arg Tyr Thr Asn Phe
130 135 140
Leu Leu Asp Thr Lys Gly Lys Leu Tyr Arg Trp Arg Ser Pro Val Ile
145 150 155 160
Ile Glu Lys Arg Gly Lys Val Glu Val Glu Gly Gln Leu Ile Asp Leu
165 170 175
Lys Arg Val Val Leu Asp Gly Ser Ala Ala Thr Pro Ile Thr Lys Val
180 185 190
Ser Ala Glu Gln Trp Gly Arg Pro
195 200
(2) INFORMATION FOR SEQ ID N0:18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1563 base pairs
(B) TYPE: Nucleic acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(ii) MOLECULE TYPE: DNA
l~
CA 02418780 2003-02-28
(vi) ORIGINAL SOURCE:
(A) Sus scrofa
ORGANISM:
(ix)FEATURE:
(A)
NAME/KEY:CDS
(B) (1) ...(603)
LOCATION:
(D) INFORMATION :wtORF5 encoding GP5 of
OTHER PRRSV
strain
IAF-94- 3182GenBank Acc ession
#U64933
(xi)SEQUENCE SEQID :
DESCRIPTION: N0:18
ATGTTGGGG AAATGCTTGACC GCGGGCTGT TGCTCGCGA TTGCCTTTT 48
MetLeuGly LysCysLeuThr AlaGlyCys CysSerArg LeuProPhe
1 5 10 15
TTGTGGTGT ATCGTGCCGTTC TGTTTTGCT GTGCTCGTC AACGCCAGC 96
LeuTrpCys IleValProPhe CysPheAla ValLeuVal AsnAlaSer
20 25 30
CCCAACAGC AGCTCCCATTTA CAGTTGATT TATAACCTG ACGATATGC 144
ProAsnSer SerSerHisLeu GlnLeuIle TyrAsnLeu ThrIleCys
35 40 45
GAGCTGAAT GGCACAGATTGG CTGAATGCA AGATTTGAT TGGGCAGTG 192
GluLeuAsn GlyThrAspTrp LeuAsnAla ArgPheAsp TrpAlaVaI
50 55 60
GAGACTTTT GTTATCTTTCCT GTGGTGACT CACATTGTC TCCTATGGT 240
GluThrPhe ValIlePhePro ValValThr HisIleVal SerTyrGly
65 70 75 80
GCCCTCACT ACCAGCCATTTT CTTGATACA GTCGGTCTG GTCACTGTG 288
AlaLeuThr ThrSerHisPhe LeuAspThr ValGlyLeu ValThrVal
85 90 95
TCTACCGCC GGGTATTACCAT GGGCGGTAT GTCTTGAGT AGCATCTAC 336
SerThrAla GlyTyrTyrHis GlyArgTyr ValLeuSer SerIleTyr
100 105 110
GCTGTCTGT GCCCTAGCTGCG TTGATTTGC TTCGTCATT AGGTTGgcg 384
AlaValCys AlaLeuAlaAla LeuIleCys PheValIle ArgLeuAla
115 120 125
AAGAATTGC ATGTCTTGGCGC TACTCATGT ACCAGATAT ACCAACTTT 432
LysAsnCys MetSerTrpArg TyrSerCys ThrArgTyr ThrAsnPhe
I30 135 140
CTTCTGGAC ACTAAGGGCAAA CTCTATCGT TGGCGGTCG CCCGTCATC 480
LeuLeuAsp ThrLysGlyLys LeuTyrArg TrpArgSer ProValIle
145 150 155 160
ATAGAGAAA AGGGGTAAAGTT GAGGTTGAA GGTCACCTG ATCGACCTC 528
IleGluLys ArgGlyLysVal GluValGlu GlyHisLeu IleAspLeu
165 170 175
AAGAGAGTT GTGCTTGACGGT TCTGCGGCA ACCCCTGTA ACCAGAGTT 576
LysArgVal ValLeuAspGly SerAlaAla ThrProVal ThrArgVal
180 185 190
TCAGCGGAA CAATGGTGTCGT CCCTAGATGACTTTTG 623
CCATGACAGC
SerAlaGlu GlnTrpCysArg Pro
195 200
18
CA 02418780 2003-02-28
ACAGCACCAC AAAAGGTGCT CTTGGCGTTT TCCATTACCT ACACGCCAGT GATGATATAT 683
GCCCTAAAGG TAAGTCGCGG CCGACTGCTA GGGCTTCTGC ACCTTTTGAT TTTCCTGAAT 743
TGTGCTTTCA CCTTCGGGTA CATGACATTC GCGCACTTTG AGAGCACAAA TAAGGTCGCG 803
CTCACTATGG GAGCAGTGGT TGCACTCCTT TGGGGGGTGT ACTCAGCCAT AGAAACCTGG 863
AAATTCATCA CCTCCAGATG CCGTTTGTGC TTGTTAGGCC GCAAGTACAT TCTGGCCCCT 923
GCCCACCACG TTGAGAGTGC CGCAGGCTTT CATCCGATTG CGGCAAGTGA TAACCACGCA 983
TTTGTCGTCC GGCGTCCCGG CTCCACTACG GTCAACGGCA CATTGGTGCC CGGGTTGAAA 1043
AGCCTCGTGT TGGGTGGCAA AAAGGCTGTC AAGCGGGGAG TGGTAAACCT CGTTAAGTAT 1103
GCCAAATAAC ACCGGCAGAC AGCAGAAGAA AAAGAAAGGG GATGGCCAGC CAGTCAATCA 1163
GCTGTGCCAG ATGCTGGGTA AGATCATCGC CCAGCAAAAC CAGTCCAGAG GTAAGGGACC 1223
GGGGAGGAAA AATAAGAAGA AAAACCCGGA GAAGCCCCAT TTTCCTCTAG CGACTGAAGA 1283
TGACGTCAGG CATCACTTTA CCCCTAGTGA GCGGCAATTG TGCCTGTCGT CAATCCAGAC 1343
AGCTTTCAAT CAAGGCGCTG GAACTTGCAC CCTGTCAGAT TCAGGGAGGA TAAGTTACGC 1403
TGTGGAGTTC AGTTTGCCTA CGCATCAGAC TGTGCGCCTG ATTCGCGTCA CAGCATCACC 1463
CTCAGCATGA TGAGCTGACA TTCCTGAAAC ATCCCAGTGT CTCAATTGGA AGAATGCGTG 1523
GTGAATGGCA CTGATTGATA TTGTGCCTCT AAGTCACCTA 1563
2) INFORMATION FOR SEQ ID N0:19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH:200 amino acids
(B) TYPE: Amino Acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Protein
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Sus scrofa
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:19:
Met Leu Gly Lys Cys Leu Thr Ala Gly Cys Cys Ser Arg Leu Pro Phe
1 5 10 15
Leu Trp Cys Ile Val Pro Phe Cys Phe Ala Val Leu Val Asn Ala Ser
20 25 30
Pro Asn Ser Ser Ser His Leu Gln Leu Ile Tyr Asn Leu Thr IIe Cys
35 40 45
Glu Leu Asn Gly Thr Asp Trp Leu Asn Ala Arg Phe Asp Trp Ala Val
50 55 60
Glu Thr Phe Val Ile Phe Pro Val Val Thr His Ile Val Ser Tyr Gly
65 70 75 BO
Ala Leu Thr Thr Ser His Phe Leu Asp Thr Val Gly Leu Val Thr Val
85 90 95
Ser Thr Ala Gly Tyr Tyr His Gly Arg Tyr Val Leu Ser Ser Ile Tyr
100 105 110
Ala Val Cys Ala Leu Ala Ala Leu Ile Cys Phe Val Ile Arg Leu Ala
115 120 125
Lys Asn Cys Met Ser Trp Arg Tyr Ser Cys Thr Arg Tyr Thr Asn Phe
130 135 140
Leu Leu Asp Thr Lys Gly Lys Leu Tyr Arg Trp Arg Ser Pro Val Ile
145 150 155 160
Ile Glu Lys Arg Gly Lys Val Glu Val Glu Gly His Leu Ile Asp Leu
165 170 175
Lys Arg Val Val Leu Asp Gly Ser Ala Ala Thr Pro Val Thr Arg Val
180 185 190
Ser Ala Glu Gln Trp Cys Arg Pro
195 200
19
CA 02418780 2003-02-28
(2) INFORMATION FOR SEQ ID N0:20:
(i)SEQUENCE
CHARACTERISTICS:
(A) LENGTH: pairs
1563 base
(B) TYPE: Nucleic acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(ii)MOLECULE DNA
TYPE:
(vi)ORIGINAL
SOURCE:
(A) ORGANISM:Sus
scrofa
(ix)FEATURE:
(A) NAME/KEY:CDS
(B) LOCATION: (1) ...(603)
(D) OTHER INFORMATION: wtORFS encodingGP5of PRRSV
strain
IAF-94-287 Accession #U6 4934
GenBank
(xi)SEQUENCE ID
DESCRIPTION: N0:20:
SEQ
ATGTTGGGG AAA TTGACC GCGGGCTGT TGCTCG CAATTGCCTTTT 48
TGC
MetLeuGly Lys LeuThr AlaGlyCys CysSer GlnLeuProPhe
Cys
1 5 10 15
TTGTGGTGT ATC CCGTTC TGTTTTGCT GCGCTC GTCAACGCCAGC 96
GTG
LeuTrpCys Ile ProPhe CysPheAla AlaLeu ValAsnAlaSer
Val
20 25 30
AACAACAGC AGC CATTTA CAGTTGATT TATAAC CTGACGATATGT 144
TCC
AsnAsnSer Ser HisLeu GlnLeuIle TyrAsn LeuThrIleCys
Ser
35 40 45
GAGCTGAAT GGC GATTGG CTGAACGAT AAATTT GACTGGGCAGTG 192
ACA
GluLeuAsn Gly AspTrp LeuAsnAsp LysPhe AspTrpAlaVal
Thr
50 55 60
GAGACTTTT GTT TTTCCT GTGTTGACT CACATT GTCTCCTATGGT 240
ATT
GluThrPhe Val PhePro ValLeuThr HisIle ValSerTyrGly
Ile
65 70 75 80
GCCCTCACC ACC CATTTC CTTGACACA GTCGGT CTGATCACTGTG 288
AGC
AlaLeuThr Thr HisPhe LeuAspThr ValGly LeuIleThrVal
Ser
85 90 95
TCTACCGCC GGA TACCAC GGGCGGTAT GTCTTG AGTAGCATCTAC 336
TAT
SerThrAla Gly TyrHis GlyArgTyr ValLeu SerSerIleTyr
Tyr
100 105 110
GCGGTCTGT GCC GCTGCG TTGATCTGC TTCGTC ATTAGGTTGGCG 384
CTG
AlaValCys Ala AlaAla LeuIleCys PheVal IleArgLeuAla
Leu
115 120 125
AAGAACTGC ATG TGGCGC TACTCGTGT ACCAGG TACACCAACTTT 432
TCC
LysAsnCys Met TrpArg TyrSerCys ThrArg TyrThrAsnPhe
Ser
130 135 140
CTCCTGGAC ACC GGCAGA CTCTATCGT TGGCGG TCACCCGTCATC 480
AAG
LeuLeuAsp Thr GlyArg LeuTyrArg TrpArg SerProValIle
Lys
CA 02418780 2003-02-28
145 150 155 160
ATA GAG AAA AAA GGT AAA GTT GAG GTC GAA GGT CAG CTG ATT GAC CTC 528
Ile Glu Lys Lys Gly Lys Val Glu Val Glu Gly Gln Leu Ile Asp Leu
165 170 175
AAG AGA GTT GTG CTT GAT GGT TCC GCG GCA ACC CCT ATA ACC AGA GTT 576
Lys Arg Val Val Leu Asp Gly Ser Ala Ala Thr Pro Ile Thr Arg Val
180 185 190
TCA GCG GAA CAA TGG GGT CGT CCC TAG ACGATTTCTG CCATGACAGC 623
Ser Ala Glu Gln Trp Gly Arg Pro
195 200
ACGGCTCCAC AAAAGGTGCT CTTGGCATTT TCCATCACCT ACACGCCAGT GATGATATAT 683
GCCCTAAAGG TAAGTCGCGG CCGACTGCTA GGGCTTTTGC ACCTTTTAAT CTTTCTGAAT 743
TGTGCTTTCA CCTTCGGGTA CATGACCTTT ACGCATTTTC AGAGCACAAA TAAGGTCGCG 803
CTCACCATGG GAGCAGTAGT CGCACTCCTT TGGGGGGTGT ACTCAGCCAT AGAAACCTGG 863
AGGTTCATCA CCTCCAGATG CCGTTTGTGC TTGTTAGGCC GCAAGTACAT TCTGGCCCCT 923
GCCCACCACG TTGAGAGTGC CGCAGGCTTT CATCCGATTG CGGCAAGTGA TAACCACGCA 983
TTTGTCGTCC GGCGTCCCGG CTCTACTACG GTTAACGGCA CATTGGTGCC CGGGTTGAAA 1043
AGCCTCGTGT TGGGTGGCAG AAAAGCTGTT AGACAGGGAG TGGTAAACCT TGTCAAGTAT 1103
GCCAAATAAC AACGGCAAGC AGCAAAAGAA AAAGAAGGGG AATGGCCAGC CAGTCAATCA 1163
GCTGTGCCAG ATGCTGGGTA AGATCATCGC CCAGCAAAAC CAGTCCAGAG GTAGGGGACC 1223
GGGGAAGAAA AATAAGAAGA AAAGCCCGGA GAAGCCCCAT TTCCCTCTAG CGACTGAAGA 1283
TGACGTCAGA CATCATTTCA CCCCTAGTGA GCGGCAATTG TGTCTGTCGT CGATCCAGAC 1343
TGCCTTCAAT CAAGGTGCTG GAACTTGTAC CCTGTCAGAT TCAGGGAGGA TAAGTTACAC 1403
TGTGGAGTTT AGTTTGCCTA CGCATCATAC CGTGCGCCTG ATTCGCGTTA CGGCATCACC 1463
TTCAGCATGA TGGGCTGGCA TTCTTGAAAC ATTCCGGTGT TTGAATTGGA AGAATGTATG 1523
GTGAATGGCA CTGATTGACA TTGTGCCTCT AAGTCACCTA 1563
2) INFORMATION FOR SEQ ID N0:21:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH:200 amino acids
(B) TYPE: Amino Acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Protein
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Sus scrofa
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:21:
Met Leu Gly Lys Cys Leu Thr Ala Gly Cys Cys Ser Gln Leu Pro Phe
1 5 10 15
Leu Trp Cys Ile Val Pro Phe Cys Phe Ala Ala Leu Val Asn Ala Ser
20 25 30
Asn Asn Ser Ser Ser His Leu Gln Leu Ile Tyr Asn Leu Thr Ile Cys
35 40 45
Glu Leu Asn Gly Thr Asp Trp Leu Asn Asp Lys Phe Asp Trp Ala Val
50 55 60
Glu Thr Phe Val Ile Phe Pro Val Leu Thr His Ile Val Ser Tyr Gly
65 70 75 80
Ala Leu Thr Thr Ser His Phe Leu Asp Thr Val Gly Leu Ile Thr Val
85 90 95
Ser Thr Ala Gly Tyr Tyr His Gly Arg Tyr Val Leu Ser Ser Ile Tyr
100 105 110
Ala Val Cys Ala Leu Ala Ala Leu Ile Cys Phe Val Ile Arg Leu Ala
21
CA 02418780 2003-02-28
115 120 125
Lys Asn Cys Met Ser Trp Arg Tyr Ser Cys Thr Arg Tyr Thr Asn Phe
130 135 140
Leu Leu Asp Thr Lys Gly Arg Leu Tyr Arg Trp Arg Ser Pro Val Ile
145 150 155 160
Ile Glu Lys Lys Gly Lys Val Glu Val Glu Gly Gln Leu Ile Asp Leu
165 170 175
Lys Arg Val Val Leu Asp Gly Ser Ala Ala Thr Pro Ile Thr Arg Val
180 185 190
Ser Ala Glu Gln Trp Gly Arg Pro
195 200
(2) INFORMATION FOR SEQ ID N0:22:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1563base rs
pai
{B) TYPE: Nucleicacid
(C) STRANDEDNESS:Single
(D) TOPOLOGY:
Linear
(ii)MOLECULE TYPE:
DNA
(vi)ORIGINAL SOURCE:
(A) ORGANISM:Susscrofa
(ix)FEATURE:
(A) NAME/KEY:CDS
(B) LOCATION: ...(603)
(1)
(D) OTHER INFORMATION: ORFSencoding PRRSV
wt GP5 strain
of
ONT-TS GenBank ssion
Acce #U64935
(xi)SEQUENCE DESCRIPTION: ID
SEQ N0:22:
ATGTTG GGG AAA TGC TTG GCG TGT TGCTCGCAA TTGCTTTTT 48
ACC GGC
MetLeu Gly Lys Cys Leu Ala Cys CysSerGln LeuLeuPhe
Thr Gly
1 5 10 15
TTGTGG TGT ATC GTG CCG TGG GTT GCGCTCGTC AGCGCCAGC 96
TCC TTT
LeuTrp Cys Ile Val Pro Trp Val AlaLeuVal SerAlaSer
Ser Phe
20 25 30
AACAGC AGC AGC TCC CAT CAG ATC TATAACTTG ACGCTATGC 144
TTA TTG
AsnSer Ser Ser Ser His Gln Ile TyrAsnLeu ThrLeuCys
Leu Leu
35 40 45
GAGCTG AAT GGC ACA GAT CTG GAT AAATTCGAT TGGGCAGTG 192
TGG GCC
GluLeu Asn Gly Thr Asp Leu Asp LysPheAsp TrpAlaVal
Trp Ala
50 55 60
GAGACT TTT GTC ATC TTT GTG ACT CACATCGTC TCCTATGGT 240
CCC TTA
GluThr Phe Val Ile Phe Val Thr HisIleVal SerTyrGly
Pro Leu
65 70 75 80
GCCCTC ACC ACC AGC CAT CTT ACA GTCGGTCTG GTTACTGTG 288
TTC GAC
AlaLeu Thr Thr Ser His Leu Thr ValGlyLeu ValThrVal
Phe Asp
85 90 95
TCTACC GCC GGT TTT CAT GGG TAT GTTCTGAGT AGCATCTAC 336
CAC CGG
SerThr Ala Gly Phe His Gly Tyr ValLeuSer SerIleTyr
His Arg
22
CA 02418780 2003-02-28
100 105 110
GCG GTC TGT GCC CTG GCT GCG TTG ATC TGT TTC GTC ATT AGG CTT GCG 384
Ala Val Cys Ala Leu Ala Ala Leu Ile Cys Phe Val Ile Arg Leu Ala
115 120 125
AAG AAC TGC ATG TCC TGG CGC TAC TCA TGT ACC AGA TAT ACC AAC TTT 432
Lys Asn Cys Met Ser Trp Arg Tyr Ser Cys Thr Arg Tyr Thr Asn Phe
130 135 140
CTT CTG GAC ACT AAG GGC AGA CTC TAT CGT TGG CGG TCG CCT GTC ATC 480
Leu Leu Asp Thr Lys Gly Arg Leu Tyr Arg Trp Arg Ser Pro Val Ile
145 150 155 160
ATA GAG AAG GGG GGT AAA GTT GAG GTC GAA GGT CAT TTG ATC GAC CTC 528
Ile Glu Lys Gly Gly Lys Val Glu Val Glu Gly His Leu Ile Asp Leu
165 170 175
AAG AGA GTT GTG CTT GAT GGT TCC GTG GCA ACC CCT ATA ACC AGA GTT 576
Lys Arg Val Val Leu Asp Gly Ser Val Ala Thr Pro Ile Thr Arg Val
180 185 190
TCA GCG GAA CAA TGG GGT CGT CCC TAG ACGACTTTTG CTATGATAGC 623
Ser Ala Glu Gln Trp Gly Arg Pro
195 200
ACGGCTCCAC AAAAGGTGAT TTTGGCGTTT TCCATCACAT ATACGCCGGT GATGATATAT 683
GCCCTAAAGG TAAGCCGCGG CCGACTGCTA GGGCTTCTGC ACCTTTTGAT TTTCCTGAAT 743
TGTGCTTTCA CCTTCGGGTA CATGACATTC ACACACTTTC AGAGCACAAA TAGGGTCGCG 803
CTCACTATGG GAGCAGTAGT TGCACTCCTT TGGGGGGTGT ACTCAGCCAT AGAGACCTGG 863
AAATTCATCA CTTCCAGATG CCGTTTGTGC TTGCTAGGCC GCAAGTACAT TCTGGCCCCT 923
GCCCACCACG TTGAAAGTGC CGCAGGCTTT CATTCGATTA CGGCAAATGA TAACCACGCA 983
TTTGTCGTCC GGCGTCCCGG CTCCACTACG GTTAACGGCA CATTGGTGCC CGGGTTGAAA 1043
AGCCTCGTGT TGGGTGGCAG AAAAGCTGTC AAACAGGGAG TGGTAAACCT TGTTAAATAT 1103
GCCAGGTAAC AACGGCAAGC AGCAAAAGAA AAAGAAGGGG GATGGCCAGC CGGTCAATCA 1163
GCTGTGCCAG ATGCTGGGTA GGATCATCGC CCAGCAGAAC CAGTCCAGAG GCAAGGGACC 1223
GGGAAAGAAA GACAAGAGGA AAAAACCGGA GAAGCCCCAT TTCCCTCTAG CGACTGAAGA 1283
TGACGTCAGG CACCACTTCA CCCCTAGTGA GCGGCAATTG TGTCTGTCGT CAATCCAAAC 1343
CGCCTTTAAT CAAGGCGCTG GAGCTTGTAT CCTGTCAGAT TCAGGGAGGA TAAGTTACAC 1403
TGTGGAGTTT AGTTTGCCAA CTCATCATAC TGTACGCCTG ATCCGCGTCA CGACACCACC 1463
TTCAGCTTGA TGGGCTGGCA TTCTTGGAGC ACCCCAGTGT TTGAATTGGA AGAGCGTGTG 1523
GTGAATGGCA CTGATTGACA TTGTGCCTCT AAGTCACCTA 1563
2) INFORMATION FOR SEQ ID N0:23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH:200 amino acids
(B) TYPE: Amino Acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Protein
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Sus scrofa
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:23:
Met Leu Gly Lys Cys Leu Thr Ala Gly Cys Cys Ser Gln Leu Leu Phe
1 5 10 15
Leu Trp Cys Ile Val Pro Ser Trp Phe Val Ala Leu Val Ser Ala Ser
23
CA 02418780 2003-02-28
20 25 30
Asn Ser Ser Ser Ser His Leu Gln Leu Ile Tyr Asn Leu Thr Leu Cys
35 40 45
Glu Leu Asn Gly Thr Asp Trp Leu Ala Asp Lys Phe Asp Trp Ala Val
50 55 60
Glu Thr Phe Val Ile Phe Pro Val Leu Thr His Ile Val Ser Tyr Gly
65 70 75 80
Ala Leu Thr Thr Ser His Phe Leu Asp Thr Val Gly Leu Val Thr Val
85 90 95
Ser Thr Ala Gly Phe His His Gly Arg Tyr Val Leu Ser Ser Ile Tyr
100 105 110
Ala Val Cys Ala Leu Ala Ala Leu Ile Cys Phe Val Ile Arg Leu Ala
115 120 125
Lys Asn Cys Met Ser Trp Arg Tyr Ser Cys Thr Arg Tyr Thr Asn Phe
130 135 140
Leu Leu Asp Thr Lys Gly Arg Leu Tyr Arg Trp Arg Ser Pro Val Ile
145 150 155 160
Ile Glu Lys Gly Gly Lys Val Glu Val Glu Gly His Leu Ile Asp Leu
165 170 175
Lys Arg Val Val Leu Asp Gly Ser Val Ala Thr Pro Ile Thr Arg Val
180 185 190
Ser Ala Glu Gln Trp Gly Arg Pro
195 200
(2) INFORMATION FOR SEQ ID N0:24:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 603 base pairs
(B) TYPE: Nucleic acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(ii) MOLECULE TYPE: DNA
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Sus scrofa
(ix) FEATURE:
(A) NAME/KEY:CDS
(B) LOCATION: (1)...(603)
(D) OTHER INFORMATION: wtORF5 encoding the GP5 of PRRSV
strain IAF-CM GenBank Accession #AF013106
(ix) FEATURE:
(A) NAME/KEY:misc feature
(B) LOCATION: 106
(C) Xaa = Ser or Tyr
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:24:
ATG TTG GGG AAA TGC TTG ACC GCG GGC TGT TGC TCG CAA TTG CCT TTT 48
Met Leu Gly Lys Cys Leu Thr Ala Gly Cys Cys Ser Gln Leu Pro Phe
1 5 10 15
TTG TGG TGT ATC GTG CCG TTC TGT TTT GCT GCG CTC GTC AAC GCC AGC 96
Leu Trp Cys Ile Val Pro Phe Cys Phe Ala Ala Leu Val Asn Ala Ser
20 25 30
AGC AGC AGC AGC TCC CAA TTG CAG TCG ATT TAT AAC CTG ACG ATA TGT 144
Ser Ser Ser Ser Ser Gln Leu Gln Ser Ile Tyr Asn Leu Thr Ile Cys
35 40 45
24
CA 02418780 2003-02-28
GAGCTG GGCACAGAT TGGCTGAAT AAA TTTGAT TGGGCAGTG 192
AAT AAT
GluLeuAsn GlyThrAsp TrpLeuAsn LysAsnPheAsp TrpAlaVal
50 55 60
GAGACTTTT GTTATCTTT CCTGTGTTG ACTCACATTGTC TCCTATGGC 240
GluThrPhe ValIlePhe ProValLeu ThrHisIleVal SerTyrGly
65 70 75 80
GCCCTCACC ACCAGCCAT TTCCTTGAC GCAGTCGGTCTG ATCACTGTG 288
AlaLeuThr ThrSerHis PheLeuAsp AlaValGlyLeu IleThrVal
85 90 95
TCTACCGCC GGATATTAC CACGGGCGG TMTGTCTTGAGT AGCGTCTAC 336
SerThrAla GlyTyrTyr HisGlyArg XaaValLeuSer SerValTyr
100 105 110
GCTGTCTGC GCCTTGGCT GCGCTGATT TGCTTCGTCATT AGGTTGACG 384
AlaValCys AlaLeuAla AlaLeuIle CysPheValIle ArgLeuThr
115 120 125
AAAAACTGC ATGTCCTGG CGCTACTCA TGTACCAGATAT ACCAACTTT 432
LysAsnCys MetSerTrp ArgTyrSer CysThrArgTyr ThrAsnPhe
130 135 140
CTTCTGGAC TCCAAGGGC AAACTCTAT CGTTGGCGGTCA CCCGTCATC 480
LeuLeuAsp SerLysGly LysLeuTyr ArgTrpArgSer ProValIle
145 150 155 160
ATAGAGAAA GGGGGTAAA GTTGAGGTT GATGGTCATCTG ATCGACCTC 528
IleGluLys GlyGIyLys ValGluVal AspGlyHisLeu IleAspLeu
165 170 175
AAGAGAGTT GTGCTTGAT GGTTCCGCG GCAACCCCTGTA ACCAAAGTT 576
LysArgVal ValLeuAsp GlySerAla AlaThrProVal ThrLysVal
180 185 190
TCAGCGGAA CAATGGTGT CGTCCCTAG 603
SerAlaGlu GlnTrpCys ArgPro
195 200
2) INFORMATION FOR SEQ ID N0:25:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH:200 amino acids
(B) TYPE: Amino Acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Protein
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Sus scrofa
(ix) FEATURE:
(A) NAME/KEY:
(B) LOCATION:
(C) Xaa = Ser or Tyr
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:25:
CA 02418780 2003-02-28
Met Leu Gly Lys Cys Leu Thr Ala Gly Cys Cys Ser Gln Leu Pro Phe
1 5 10 15
Leu Trp Cys Ile Val Pro Phe Cys Phe Ala Ala Leu Val Asn Ala Ser
20 25 30
Ser Ser Ser Ser Ser Gln Leu Gln Ser Ile Tyr Asn Leu Thr Ile Cys
35 40 45
Glu Leu Asn Gly Thr Asp Trp Leu Asn Lys Asn Phe Asp Trp Ala Val
50 55 60
Glu Thr Phe Val Ile Phe Pro Val Leu Thr His Ile Val Ser Tyr Gly
65 70 75 80
Ala Leu Thr Thr Ser His Phe Leu Asp Ala Val Gly Leu Ile Thr Val
85 90 95
Ser Thr Ala Gly Tyr Tyr His Gly Arg Xaa Val Leu Ser Ser Val Tyr
100 105 110
Ala Val Cys Ala Leu Ala Ala Leu Ile Cys Phe Val Ile Arg Leu Thr
115 120 125
Lys Asn Cys Met Ser Trp Arg Tyr Ser Cys Thr Arg Tyr Thr Asn Phe
130 135 140
Leu Leu Asp Ser Lys Gly Lys Leu Tyr Arg Trp Arg Ser Pro Val Ile
145 150 155 160
Ile Glu Lys Gly Gly Lys Val Glu Val Asp Gly His Leu Ile Asp Leu
165 170 175
Lys Arg Val Val Leu Asp Gly Ser Ala Ala Thr Pro Val Thr Lys Val
180 185 190
Ser Ala Glu Gln Trp Cys Arg Pro
195 200
(2) INFORMATION FOR SEQ ID N0:26:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15411 base pairs
(B) TYPE: Nucleic acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(ii) MOLECULE TYPE: DNA
(vi) ORIGINAL SOURCE:
(A) ORGANISM:Sus scrofa
(ix) FEATURE:
(A) NAME/KEY:CDS
(B) LOCATION: (13788)...(14390)
(D) OTHER INFORMATION: wtORF5 encoding GP5 of PRRSV strain
VR-2332 GenBank Accession #U87392
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:26:
ATGACGTATA GGTGTTGGCT CTATGCCTTG GCATTTGTAT TGTCAGGAGC TGTGACCATT 60
GGCACAGCCC AAAACTTGCT GCACAGAAAC ACCCTTCTGT GATAGCCTCC TTCAGGGGAG 120
CTTAGGGTTT GTCCCTAGCA CCTTGCTTCC GGAGTTGCAC TGCTTTACGG TCTCTCCACC 180
CCTTTAACCA TGTCTGGGAT ACTTGATCGG TGCACGTGTA CCCCCAATGC CAGGGTGTTT 240
ATGGCGGAGG GCCAAGTCTA CTGCACACGA TGCCTCAGTG CACGGTCTCT CCTTCCCCTG 300
AACCTCCAAG TTTCTGAGCT CGGGGTGCTA GGCCTATTCT ACAGGCCCGA AGAGCCACTC 360
CGGTGGACGT TGCCACGTGC ATTCCCCACT GTTGAGTGCT CCCCCGCCGG GGCCTGCTGG 420
CTTTCTGCAA TCTTTCCAAT CGCACGAATG ACCAGTGGAA ACCTGAACTT CCAACAAAGA 480
ATGGTACGGG TCGCAGCTGA GCTTTACAGA GCCGGCCAGC TCACCCCTGC AGTCTTGAAG 540
GCTCTACAAG TTTATGAACG GGGTTGCCGC TGGTACCCCA TTGTTGGACC TGTCCCTGGA 600
GTGGCCGTTT TCGCCAATTC CCTACATGTG AGTGATAAAC CTTTCCCGGG AGCAACTCAC 660
26
CA 02418780 2003-02-28
GTGTTGACCA ACCTGCCGCT CCCGCAGAGA CCCAAGCCTG AAGACTTTTG CCCCTTTGAG 720
TGTGCTATGG CTACTGTCTA TGACATTGGT CATGACGCCG TCATGTATGT GGCCGAAAGG 780
AAAGTCTCCT GGGCCCCTCG TGGCGGGGAT GAAGTGAAAT TTGAAGCTGT CCCCGGGGAG 840
TTGAAGTTGA TTGCGAACCG GCTCCGCACC TCCTTCCCGC CCCACCACAC AGTGGACATG 900
TCTAAGTTCG CCTTCACAGC CCCTGGGTGT GGTGTTTCTA TGCGGGTCGA ACGCCAACAC 960
GGCTGCCTTC CCGCTGACAC TGTCCCTGAA GGCAACTGCT GGTGGAGCTT GTTTGACTTG 1020
CTTCCACTGG AAGTTCAGAA CAAAGAAATT CGCCATGCTA ACCAATTTGG CTACCAGACC 1080
AAGCATGGTG TCTCTGGCAA GTACCTACAG CGGAGGCTGC AAGTTAATGG TCTCCGAGCA 1140
GTAACTGACC TAAACGGACC TATCGTCGTA CAGTACTTCT CCGTTAAGGA GAGTTGGATC 1200
CGCCATTTGA AACTGGCGGG AGAACCCAGC TACTCTGGGT TTGAGGACCT CCTCAGAATA 1260
AGGGTTGAGC CTAACACGTC GCCATTGGCT GACAAGGAAG AAAAAATTTT CCGGTTTGGC 1320
AGTCACAAGT GGTACGGCGC TGGAAAGAGA GCAAGAAAAG CACGCTCTTG TGCGACTGCT 1380
ACAGTCGCTG GCCGCGCTTT GTCCGTTCGT GAAACCCGGC AGGCCAAGGA GCACGAGGTT 1440
GCCGGCGCCA ACAAGGCTGA GCACCTCAAA CACTACTCCC CGCCTGCCGA AGGGAATTGT 1500
GGTTGGCACT GCATTTCCGC CATCGCCAAC CGGATGGTGA ATTCCAAATT TGAAACCACC 1560
CTTCCCGAAA GAGTGAGACC TCCAGATGAC TGGGCTACTG ACGAGGATCT TGTGAATGCC 1620
ATCCAAATCC TCAGACTCCC TGCGGCCTTA GACAGGAACG GTGCTTGTAC TAGCGCCAAG 1680
TACGTACTTA AGCTGGAAGG TGAGCATTGG ACTGTCACTG TGACCCCTGG GATGTCCCCT 1740
TCTTTGCTCC CTCTTGAATG TGTTCAGGGC TGTTGTGGGC ACAAGGGCGG TCTTGGTTCC 1800
CCAGATGCAG TCGAGGTCTC CGGATTTGAC CCTGCCTGCC TTGACCGGCT GGCTGAGGTG 1860
ATGCACCTGC CTAGCAGTGC TATCCCAGCC GCTCTGGCCG AAATGTCTGG CGATTCCGAT 1920
CGTTCGGCTT CTCCGGTCAC CACCGTGTGG ACTGTTTCGC AGTTCTTTGC CCGTCACAGC 1980
GGAGGGAATC ACCCTGACCA AGTGCGCTTA GGGAAAATTA TCAGCCTTTG TCAGGTGATT 2040
GAGGACTGCT GCTGTTCCCA GAACAAAACC AACCGGGTCA CCCCGGAGGA GGTCGCAGCA 2100
AAGATTGACC TGTACCTCCG TGGTGCAACA AATCTTGAAG AATGCTTGGC CAGGCTTGAG 2160
AAAGCGCGCC CGCCACGCGT AATCGACACC TCCTTTGATT GGGATGTTGT GCTCCCTGGG 2220
GTTGAGGCGG CAACCCAGAC GATCAAGCTG CCCCAGGTCA ACCAGTGTCG TGCTCTGGTC 2280
CCTGTTGTGA CTCAAAAGTC CTTGGACAAC AACTCGGTCC CCCTGACCGC CTTTTCACTG 2340
GCTAACTACT ACTACCGTGC GCAAGGTGAC GAAGTTCGTC ACCGTGAAAG ACTAACCGCC 2400
GTGCTCTCCA AGTTGGAAAA GGTTGTTCGA GAAGAATATG GGCTCATGCC AACCGAGCCT 2460
GGTCCACGGC CCACACTGCC ACGCGGGCTC GACGAACTCA AAGACCAGAT GGAGGAGGAC 2520
TTGCTGAAAC TGGCTAACGC CCAGACGACT TCGGACATGA TGGCCTGGGC AGTCGAGCAG 2580
GTTGACCTAA AAACTTGGGT CAAGAACTAC CCGCGGTGGA CACCACCACC CCCTCCGCCA 2640
AAAGTTCAGC CTCGAAAAAC GAAGCCTGTC AAGAGCTTGC CGGAGAGAAA GCCTGTCCCC 2700
GCCCCGCGCA GGAAGGTTGG GTCCGATTGT GGCAGCCCGG TTTCATTAGG CGGCGATGTC 2760
CCTAACAGTT GGGAAGATTT GGCTGTTAGT AGCCCCTTTG ATCTCCCGAC CCCACCTGAG 2820
CCGGCAACAC CTTCAAGTGA GCTGGTGATT GTGTCCTCAC CGCAATGCAT CTTCAGGCCG 2880
GCGACACCCT TGAGTGAGCC GGCTCCAATT CCCGCACCTC GCGGAACTGT GTCTCGACCG 2940
GTGACACCCT TGAGTGAGCC GATCCCTGTG CCCGCACCGC GGCGTAAGTT TCAGCAGGTG 3000
AAAAGATTGA GTTCGGCGGC GGCAATCCCA CCGTACCAGG ACGAGCCCCT GGATTTGTCT 3060
GCTTCCTCAC AGACTGAATA TGAGGCCTCT CCCCCAGCAC CGCCGCAGAG CGGGGGCGTT 3120
CTGGGAGTAG AGGGGCATGA AGCTGAGGAA ACCCTGAGTG AAATCTCGGA CATGTCGGGT 3180
AACATTAAAC CTGCGTCCGT GTCATCAAGC AGCTCCTTGT CCAGCGTGAG AATCACACGC 3240
CCAAAATACT CAGCTCAAGC CATCATCGAC TCGGGCGGGC CCTGCAGTGG GCATCTCCAA 3300
GAGGTAAAGG AAACATGCCT TAGTGTCATG CGCGAGGCAT GTGATGCGAC TAAGCTTGAT 3360
GACCCTGCTA CGCAGGAATG GCTTTCTCGC ATGTGGGATC GGGTGGACAT GCTGACTTGG 3420
CGCAACACGT CTGTTTACCA GGCGATTTGC ACCTTAGATG GCAGGTTAAA GTTCCTCCCA 3480
AAAATGATAC TCGAGACACC GCCGCCCTAT CCGTGTGAGT TTGTGATGAT GCCTCACACG 3540
CCTGCACCTT CCGTAGGTGC GGAGAGCGAC CTTACCATTG GCTCAGTTGC TACTGAAGAT 3600
GTTCCACGCA TCCTCGAGAA AATAGAAAAT GTCGGCGAGA TGGCCAACCA GGGACCCTTG 3660
GCCTTCTCCG AGGATAAACC GGTAGATGAC CAACTTGTCA ACGACCCCCG GATATCGTCG 3720
CGGAGGCCTG ACGAGAGCAC ATCAGCTCCG TCCGCAGGCA CAGGTGGCGC CGGCTCTTTT 3780
ACCGATTTGC CGCCTTCAGA TGGCGCGGAT GCGGACGGGG GGGGGCCGTT TCGGACGGTA 3840
AAAAGAAAAG CTGAAAGGCT CTTTGACCAA CTGAGCCGTC AGGTTTTTGA CCTCGTCTCC 3900
CATCTCCCTG TTTTCTTCTC ACGCCTTTTC TACCCTGGCG GTGGTTATTC TCCGGGTGAT 3960
TGGGGTTTTG CAGCTTTTAC TCTATTGTGC CTCTTTTTAT GTTACAGTTA CCCAGCCTTT 4020
GGTATTGCTC CCCTCTTGGG TGTGTTTTCT GGGTCTTCTC GGCGCGTTCG AATGGGGGTT 4080
TTTGGCTGCT GGTTGGCTTT TGCTGTTGGT CTGTTCAAGC CTGTGTCCGA CCCAGTCGGC 4140
GCTGCTTGTG AGTTTGACTC GCCAGAGTGT AGAAACATCC TTCATTCTTT TGAGCTTCTC 4200
AAACCTTGGG ACCCTGTTCG CAGCCTTGTT GTGGGCCCCG TCGGTCTCGG TCTTGCCATT 4260
CTTGGCAGGT TACTGGGCGG GGCACGCTGC ATCTGGCACT TTTTGCTTAG GCTTGGCATT 4320
27
CA 02418780 2003-02-28
GTTGCAGACT GTATCTTGGC TGGAGCTTAC GTGCTTTCTC AAGGTAGGTG TAAAAAGTGC 4380
TGGGGATCTT GTATAAGAAC TGCTCCTAAT GAGGTCGCTT TTAACGTGTT TCCTTTCACA 4440
CGTGCGACCA GGTCGTCACT TATCGACCTG TGCGATCGGT 'L'TTGTGCGCC AAAAGGAATG 4500
GACCCCATTT TTCTCGCCAC TGGGTGGCGC GGGTGCTGGG CCGGCCGAAG CCCCATTGAG 4560
CAACCCTCTG AAAAACCCAT CGCGTTTGCC CAATTGGATG AAAAGAAGAT TACGGCTAGG 4620
ACTGTGGTCG CCCAGCCTTA TGACCCCAAC CAAGCCGTAA AGTGCTTGCG GGTATTGCAG 4680
TCGGGTGGGG CGATGGTGGC TAAGGCGGTC CCAAAAGTGG TCAAGGTTTC CGCTGTTCCA 4740
TTCCGAGCCC CCTTCTTTCC CACTGGAGTG AAAGTTGACC CTGATTGCAG GGTCGTGGTT 4800
GACCCTGACA CTTTCACTGC AGCTCTCCGG TCTGGCTACT CCACCACAAA CCTCGTCCTT 4860
GGTGTAGGGG ACTTTGCCCA GCTGAATGGA TTAAAAATCA GGCAAATTTC CAAGCCTTCA 4920
GGGGGAGGCC CACATCTCAT GGCTGCCCTG CATGTTGCCT GCTCGATGGC TCTGCACATG 4980
CTTGCTGGGA TTTATGTGAC TGCGGTGGGT TCTTGCGGCA CCGGCACCAA CGACCCGTGG 5040
TGCGCTAACC CGTTTGCCGT CCCTGGCTAC GGACCTGGCT CTCTCTGCAC GTCCAGGTTG 5100
TGCATTTCCC AACACGGCCT TACCCTGCCC TTGACAGCAC TTGTGGCGGG ATTCGGTATT 5160
CAAGAAATTG CCTTGGTCGT TTTGATTTTT GTTTCCATCG GAGGCATGGC TCATAGGTTG 5220
AGCTGTAAGG CTGACATGCT GTGTGTTTTG CTTGCAATTG CCAGCTATGT TTGGGTACCT 5280
CTTACCTGGT TGCTTTGTGT GTTTCCTTGC TGGTTGCGCT GTTTTTCTTT GCACCCCCTC 5340
ACCATCCTAT GGTTGGTGTT TTTCTTGATT TCTGTGAATA TGCCTTCAGG AATCTTGGCC 5400
ATGGTGTTGT TGGTTTCTCT TTGGCTTCTT GGTCGTTATA CTAATGTTGC TGGCCTTGTC 5460
ACCCCCTACG ACATTCATCA TTACACCAGT GGCCCCCGCG GTGTTGCCGC CTTGGCTACC 5520
GCACCAGATG GGACCTACTT GGCCGCTGTC CGCCGCGCTG CGTTGACTGG CCGCACCATG 5580
CTGTTTACCC CGTCCCAGCT TGGGTCTCTT CTTGAGGGTG CTTTCAGAAC TCGAAAGCCC 5640
TCACTGAACA CCGTCAATGT GATCGGGTCC TCCATGGGCT CTGGCGGGGT GTTTACCATC 5700
GACGGGAAAG TCAAGTGCGT AACTGCCGCA CATGTCCTTA CGGGCAATTC AGCTCGGGTT 5760
TCCGGGGTCG GCTTCAATCA AATGCTTGAC TTTGACGTAA AGGGAGATTT CGCTATAGCT 5820
GATTGCCCGA ATTGGCAAGG GGCTGCCCCC AAGACCCAAT TCTGCACGGA TGGATGGACT 5880
GGCCGTGCCT ATTGGCTAAC ATCCTCTGGC GTCGAACCCG GCGTCATTGG AAAAGGATTC 5940
GCCTTCTGCT TCACCGCATG TGGCGATTCC GGGTCCCCAG TGATCACCGA GGCCGGTGAG 6000
CTTGTCGGCG TTCACACGGG ATCGAATAAA CAAGGGGGGG GCATTGTTAC GCGCCCCTCA 6060
GGCCAGTTTT GTAATGTGGC ACCCATCAAG CTAAGCGAAT TAAGTGAATT CTTTGCTGGG 6120
CCTAAGGTCC CGCTCGGTGA TGTGAAGGTC GGCAGCCACA TAATTAAAGA CATAAGCGAG 6180
GTGCCTTCAG ATCTTTGTGC CTTGCTTGCT GCCAAACCTG AACTGGAAGG AGGCCTCTCC 6240
ACCGTCCAAC TTCTTTGTGT GTTTTTTCTC CTGTGGAGAA TGATGGGACA TGCCTGGACG 6300
CCCTTGGTTG CTGTGAGTTT CTTTATTTTG AATGAGGTTC TCCCAGCCGT CCTGGTCCGG 6360
AGTGTTTTCT CCTTTGGAAT GTTTGTGCTA TCCTGGCTCA CGCCATGGTC TGCGCAAGTT 6420
CTGATGATCA GGCTTCTGAC AGCAGCTCTT AACAGGAACA GATGGTCACT TGCCTTTTTC 6480
AGCCTCGGTG CAGTGACCGG TTTTGTCGCA GATCTTGCGG CCACTCAGGG GCATCCGTTG 6540
CAGGCAGTGA TGAATTTGAG CACCTATGCA TTCCTGCCTC GGATGATGGT TGTGACCTCA 6600
CCAGTCCCAG TGATCACGTG TGGTGTCGTG CACCTACTTG CCATCATTTT GTACTTGTTT 6660
AAGTACCGTG GCCCGCACCA TATCCTTGTT GGCGATGGAG TGTTCTCTGC GGCTTTCTTC 6720
TTGAGATACT TTGCCGAGGG AAAGTTGAGG GAAGGGGTGT CGCAATCCTG CGGAATGAAT 6780
CATGAGTCTC TGACTGGTGC CCTCGCTATG AGACTCAATG ACGAGGACTT GGATTTCCTT 6840
ATGAAATGGA CTGATTTTAA GTGCTTTGTT TCTGCGTCCA ACATGAGGAA TGCAGCGGGT 6900
CAATTTATCG AGGCTGCCTA TGCTAAAGCA CTTAGAGTAG AACTGGCCCA GTTGGTGCAG 6960
GTTGATAAAG TTCGAGGTAC TTTGGCCAAA CTTGAAGCTT TTGCTGATAC CGTGGCACCT 7020
CAACTCTCGC CCGGTGACAT TGTTGTCGCT CTCGGCCACA CGCCTGTTGG CAGTATCTTC 7080
GACCTAAAGG TTGGTAGCAC CAAGCATACC CTCCAAGCCA TTGAGACCAG AGTCCTTGCT 7140
GGGTCCAAAA TGACCGTGGC GCGCGTCGTC GACCCGACCC CCACGCCCCC ACCCGCACCC 7200
GTGCCCATCC CCCTCCCACC GAAAGTTCTG GAGAATGGCC CCAACGCTTG GGGGGATGAG 7260
GACCGTTTGA ATAAGAAGAA GAGGCGCAGG ATGGAAGCCC TCGGCATCTA TGTTATGGGC 7320
GGGAAAAAGT ACCAGAAATT TTGGGACAAG AATTCCGGTG ATGTGTTTTA TGAGGAGGTC 7380
CATAATAACA CAGATGAGTG GGAGTGTCTC AGAGTTGGCG ACCCTGCCGA CTTTGACCCT 7440
GAGAAGGGAA CTCTGTGTGG ACATGTCACC ATTGAAAACA AGGCTTACCA TGTTTACACC 7500
TCCCCATCTG GTAAGAAGTT CTTGGTCCCC GTCAACCCAG AGAATGGAAG AGTTCAATGG 7560
GAAGCTGCAA AGCTTTCCGT GGAGCAGGCC CTAGGTATGA TGAATGTCGA CGGCGAACTG 7620
ACTGCCAAAG AACTGGAGAA ACTGAAAAGA ATAATTGACA AACTCCAGGG CCTGACTAAG 7680
GAGCAGTGTT TAAACTGCTA GCCGCCAGCG ACTTGACCCG CTGTGGTCGC GGCGGCTTGG 7740
TTGTTACTGA AACAGCGGTA AAAATAGTCA AATTTCACAA CCGGACCTTC ACCCTGGGAC 7800
CTGTGAATTT AAAAGTGGCC AGTGAGGTTG AGCTAAAAGA CGCGGTTGAG CACAACCAAC 7860
ACCCGGTTGC GAGACCGATC GATGGTGGAG TTGTGCTCCT GCGTTCCGCG GTTCCTTCGC 7920
TTATAGACGT CTTGATCTCC GGTGCTGATG CATCTCCCAA GTTACTTGCC CATCACGGGC 7980
7g
CA 02418780 2003-02-28
CGGGAAACAC TGGGATCGAT GGCACGCTCT GGGATTTTGA GTCCGAAGCC ACTAAAGAGG 8040
AAGTCGCACT CAGTGCGCAA ATAATACAGG CTTGTGACAT TAGGCGCGGC GACGCTCCTG 8100
AAATTGGTCT CCCTTACAAG CTGTACCCTG TTAGGGGTAA CCCTGAGCGG GTGAAAGGAG 8160
TTCTGCAGAA TACAAGGTTT GGAGACATAC CTTACAAAAC CCCCAGTGAC ACTGGAAGCC 8220
CAGTGCACGC GGCTGCCTGC CTTACGCCCA ACGCCACTCC GGTGACTGAT GGGCGCTCCG 8280
TCTTGGCCAC GACCATGCCC CCCGGGTTTG AGTTATATGT ACCGACCATA CCAGCGTCTG 8340
TCCTTGATTA CCTTGACTCT AGGCCTGACT GCCCTAAACA GCTGACAGAG CACGGCTGCG 8400
AAGATGCCGC ACTGAAAGAC CTCTCTAAAT ATGACTTGTC CACCCAAGGC TTTGTTTTAC 8460
CTGGAGTTCT TCGCCTTGTG CGGAAATACC TGTTTGCCCA TGTAGGTAAG TGCCCACCCG 8520
TTCATCGGCC TTCTACTTAC CCTGCTAAGA ATTCTATGGC TGGAATAAAT GGGAACAGGT 8580
TCCCAACCAA GGACATTCAG AGCGTCCCTG AAATCGACGT TCTGTGCGCA CAGGCTGTGC 8640
GAGAAAACTG GCAAACTGTC ACCCCTTGTA CTCTTAAGAA ACAGTATTGC GGGAAGAAGA 8700
AGACTAGGAC CATACTCGGC ACCAATAACT TCATCGCACT AGCCCACCGA GCAGTGTTGA 8760
GTGGTGTTAC CCAGGGCTTC ATGAAAAAGG CGTTTAACTC GCCCATCGCC CTCGGAAAGA 8820
ACAAGTTTAA GGAGCTACAG ACTCCGGTCC TGGGCAGGTG CCTTGAAGCT GATCTCGCAT 8880
CCTGCGATCG ATCCACGCCT GCAATTGTCC GCTGGTTTGC CGCCAACCTT CTTTATGAAC 8940
TTGCCTGTGC TGAAGAGCAT CTACCGTCGT ACGTGCTGAA CTGCTGCCAC GACTTACTGG 9000
TCACGCAGTC CGGCGCAGTG ACTAAGAGAG GTGGCCTGTC GTCTGGCGAC CCGATCACCT 9060
CTGTGTCTAA CACCATTTAT AGTTTGGTGA TCTATGCACA GCATATGGTG CTTAGTTACT 9120
TCAAAAGTGG TCACCCCCAT GGCCTTCTGT TCTTACAAGA CCAGCTAAAG TTTGAGGACA 9180
TGCTCAAGGT TCAACCCCTG ATCGTCTATT CGGACGACCT CGTGCTGTAT GCCGAGTCTC 9240
CCACCATGCC AAACTATCAC TGGTGGGTTG AACATCTGAA TTTGATGCTG GGGTTTCAGA 9300
CGGACCCAAA GAAGACAGCA ATAACAGACT CGCCATCATT TCTAGGCTGT AGAATAATAA 9360
ATGGGCGCCA GCTAGTCCCC AACCGTGACA GGATCCTCGC GGCCCTCGCC TATCACATGA 9420
AGGCGAGTAA TGTTTCTGAA TACTATGCCT CAGCGGCTGC AATACTCATG GACAGCTGTG 9480
CTTGTTTGGA GTATGATCCT GAATGGTTTG AAGAACTTGT AGTTGGAATA GCGCAGTGCG 9540
CCCGCAAGGA CGGCTACAGC TTTCCCGGCA CGCCGTTCTT CATGTCCATG TGGGAAAAAC 9600
TCAGGTCCAA TTATGAGGGG AAGAAGTCGA GAGTGTGCGG GTACTGCGGG GCCCCGGCCC 9660
CGTACGCTAC TGCCTGTGGC CTCGACGTCT GCATTTACCA CACCCACTTC CACCAGCATT 9720
GTCCAGTCAC AATCTGGTGT GGCCATCCAG CGGGTTCTGG TTCTTGTAGT GAGTGCAAAT 9780
CCCCTGTAGG GAAAGGCACA AGCCCTTTAG ACGAGGTGCT GGAACAAGTC CCGTATAAGC 9840
CCCCACGGAC CGTTATCATG CATGTGGAGC AGGGTCTCAC CCCCCTTGAT CCAGGTAGAT 9900
ACCAAACTCG CCGCGGATTA GTCTCTGTCA GGCGTGGAAT TAGGGGAAAT GAAGTTGGAC 9960
TACCAGACGG TGATTATGCT AGCACCGCCT TGCTCCCTAC CTGCAAAGAG ATCAACATGG 10020
TCGCTGTCGC TTCCAATGTA TTGCGCAGCA GGTTCATCAT CGGCCCACCC GGTGCTGGGA 10080
AAACATACTG GCTCCTTCAA CAGGTCCAGG ATGGTGATGT TATTTACACA CCAACTCACC 10140
AGACCATGCT TGACATGATT AGGGCTTTGG GGACGTGCCG GTTCAACGTC CCGGCAGGCA 10200
CAACGCTGCA ATTCCCCGTC CCCTCCCGCA CCGGTCCGTG GGTTCGCATC CTAGCCGGCG 10260
GTTGGTGTCC TGGCAAGAAT TCCTTCCTAG ATGAAGCAGC GTATTGCAAT CACCTTGATG 10320
TTTTGAGGCT TCTTAGTAAA ACTACCCTCA CCTGTCTAGG AGACTTCAAG CAACTCCACC 10380
CAGTGGGTTT TGATTCTCAT TGCTATGTTT TTGACATCAT GCCTCAAACT CAACTGAAGA 10440
CCATCTGGAG GTTTGGACAG AATATCTGTG ATGCCATTCA GCCAGATTAC AGGGACAAAC 10500
TCATGTCCAT GGTCAACACA ACCCGTGTGA CCTACGTGGA AAAACCTGTC AGGTATGGGC 10560
AGGTCCTCAC CCCCTACCAC AGGGACCGAG AGGACGACGC CATCACTATT GACTCCAGTC 10620
AAGGCGCCAC ATTCGATGTG GTTACATTGC ATTTGCCCAC TAAAGATTCA CTCAACAGGC 10680
AAAGAGCCCT TGTTGCTATC ACCAGGGCAA GACACGCTAT CTTTGTGTAT GACCCACACA 10740
GGCAGCTGCA GGGCTTGTTT GATCTTCCTG CAAAAGGCAC GCCCGTCAAC CTCGCAGTGC 10800
ACTGCGACGG GCAGCTGATC GTGCTGGATA GAAATAACAA AGAATGCACG GTTGCTCAGG 10860
CTCTAGGCAA CGGGGATAAA TTTAGGGCCA CAGACAAGCG TGTTGTAGAT TCTCTCCGCG 10920
CCATTTGTGC TGATCTAGAA GGGTCGAGCT CTCCGCTCCC CAAGGTCGCA CACAACTTGG 10980
GATTTTATTT CTCACCTGAT TTAACACAGT TTGCTAAACT CCCAGTAGAA CTTGCACCTC 11040
ACTGGCCCGT GGTGTCAACC CAGAACAATG AAAAGTGGCC GGATCGGCTG GTTGCCAGCC 11100
TTCGCCCTAT CCATAAATAC AGCCGCGCGT GCATCGGTGC CGGCTATATG GTGGGCCCTT 11160
CGGTGTTTCT AGGCACTCCT GGGGTCGTGT CATACTATCT CACAAAATTT GTTAAGGGCG 11220
GGGCTCAAGT GCTTCCGGAG ACGGTTTTCA GCACCGGCCG AATTGAGGTA GACTGCCGGG 11280
AATATCTTGA TGATCGGGAG CGAGAAGTTG CTGCGTCCCT CCCACACGGT TTCATTGGCG 11340
ACGTCAAAGG CACTACCGTT GGAGGATGTC ATCATGTCAC CTCCAGATAC CTCCCGCGCG 11400
TCCTTCCCAA GGAATCAGTT GCGGTAGTCG GGGTTTCAAG CCCCGGAAAA GCCGCGAAAG 11460
CATTGTGCAC ACTGACAGAT GTGTACCTCC CAGATCTTGA AGCCTATCTC CACCCGGAGA 11520
CCCAGTCCAA GTGCTGGAAA ATGATGTTGG ACTTCAAAGA AGTTCGACTA ATGGTCTGGA 11580
AAGACAAAAC AGCCTATTTC CAACTTGAAG GTCGCTATTT CACCTGGTAT CAGCTTGCCA 11640
29
CA 02418780 2003-02-28
GCTATGCCTC GTACATCCGT GTTCCCGTCA ACTCTACGGT GTACTTGGAC CCCTGCATGG 11700
GCCCCGCCCT TTGCAACAGG AGAGTCGTCG GGTCCACCCA CTGGGGGGCT GACCTCGCGG 11760
TCACCCCTTA TGATTACGGC GCTAAAATTA TCCTGTCTAG CGCGTACCAT GGTGAAATGC 11820
CCCCCGGATA CAAAATTCTG GCGTGCGCGG AGTTCTCGTT GGATGACCCA GTTAAGTACA 11880
AACATACCTG GGGGTTTGAA TCGGATACAG CGTATCTGTA TGAGTTCACC GGAAACGGTG 11940
AGGACTGGGA GGATTACAAT GATGCGTTTC GTGCGCGCCA GGAAGGGAAA ATTTATAAGG 12000
CCACTGCCAC CAGCTTGAAG TTTTATTTTC CCCCGGGCCC TGTCATTGAA CCAACTTTAG 12060
GCCTGAATTG AAATGAAATG GGGTCCATGC AAAGCCTTTT TGACAAAATT GGCCAACTTT 12120
TTGTGGATGC TTTCACGGAG TTCTTGGTGT CCATTGTTGA TATCATTATA TTTTTGGCCA 12180
TTTTGTTTGG CTTCACCATC GCCGGTTGGC TGGTGGTCTT TTGCATCAGA TTGGTTTGCT 12240
CCGCGATACT CCGTACGCGC CCTGCCATTC ACTCTGAGCA ATTACAGAAG ATCTTATGAG 12300
GCCTTTCTTT CCCAGTGCCA AGTGGACATT CCCACCTGGG GAACTAAACA TCCTTTGGGG 12360
ATGCTTTGGC ACCATAAGGT GTCAACCCTG ATTGATGAAA TGGTGTCGCG TCGAATGTAC 12420
CGCATCATGG AAAAAGCAGG GCAGGCTGCC TGGAAACAGG TGGTGAGCGA GGCTACGCTG 12480
TCTCGCATTA GTAGTTTGGA TGTGGTGGCT CATTTTCAGC ATCTAGCCGC CATTGAAGCC 12540
GAGACCTGTA AATATTTGGC CTCCCGGCTG CCCATGCTAC ACAACCTGCG CATGACAGGG 12600
TCAAATGTAA CCATAGTGTA TAATAGCACT TTGAATCAGG TGTTTGCTAT TTTTCCAACC 12660
CCTGGTTCCC GGCCAAAGCT TCATGATTTT CAGCAATGGT TAATAGCTGT ACATTCCTCC 12720
ATATTTTCCT CTGTTGCAGC TTCTTGTACT CTTTTTGTTG TGCTGTGGTT GCGGGTTCCA 12780
ATACTACGTA CTGTTTTTGG TTTCCGCTGG TTAGGGGCAA TTTTTCTTTC GAACTCACAG 12840
TGAATTACAC GGTGTGTCCA CCTTGCCTCA CCCGGCAAGC AGCCACAGAG ATCTACGAAC 12900
CCGGTAGGTC TCTTTGGTGC AGGATAGGGT ATGACCGATG TGGGGAGGAC GATCATGACG 12960
AGCTAGGGTT TATGATACCG CCTGGCCTCT CCAGCGAAGG CCACTTGACT GGTGTTTACG 13020
CCTGGTTGGC GTTCTTGTCC TTCAGCTACA CGGCCCAGTT CCATCCCGAG ATATTCGGGA 13080
TAGGGAATGT GAGTCGAGTT TATGTTGACA TCAAACATCA ACTCATCTGC GCCGAACATG 13140
ACGGGCAGAA CACCACCTTG CCTCGTCATG ACAACATTTC AGCCGTGTTT CAGACCTATT 13200
ACCAACATCA AGTCGACGGC GGCAATTGGT TTCACCTAGA ATGGCTTCGT CCCTTCTTTT 13260
CCTCGTGGTT GGTTTTAAAT GTCTCTTGGT TTCTCAGGCG TTCGCCTGCA AACCATGTTT 13320
CAGTTCGAGT CTTGCAGATA TTAAGACCAA CACCACCGCA GCGGCAAGCT TTGCTGTCCT 13380
CCAAGACATC AGTTGCCTTA GGCATCGCGA CTCGGCCTCT GAGGCGATTC GCAAAATCCC 13440
TCAGTGCCGT ACGGCGATAG GGACACCCGT GTATGTTACC ATCACAGCCA ATGTGACAGA 13500
TGAGAATTAT TTACATTCTT CTGATCTCCT CATGCTTTCT TCTTGCCTTT TCTATGCTTC 13560
TGAGATGAGT GAAAAGGGAT TTAAGGTGGT ATTTGGCAAT GTGTCAGGCA TCGTGGCTGT 13620
GTGTGTCAAT TTTACCAGCT ACGTCCAACA TGTCAAGGAG TTTACCCAAC GCTCCCTGGT 13680
GGTCGACCAT GTGCGGTTGC TCCATTTCAT GACACCTGAG ACCATGAGGT GGGCAACTGT 13740
TTTAGCCTGT CTTTTTGCCA TTCTGTTGGC AATTTGAATG TTTAAGT ATG TTG GAG 13796
Met Leu Glu
1
AAATGCTTGACC GCGGGCTGT TGCTCGCGA TTGCTTTCTTTG TGGTGT 13844
LysCysLeuThr AlaGlyCys CysSerArg LeuLeuSerLeu TrpCys
10 15
ATCGTGCCGTTC TGTTTTGCT GTGCTCGCC AACGCCAGCAAC GACAGC 13892
IleValProPhe CysPheAla ValLeuAla AsnAlaSerAsn AspSer
20 25 30 35
AGCTCCCATCTA CAGCTGATT TACAACTTG ACGCTATGTGAG CTGAAT 13940
SerSerHisLeu GlnLeuIle TyrAsnLeu ThrLeuCysGlu LeuAsn
40 45 50
GGCACAGATTGG CTAGCTAAC AAATTTGAT TGGGCAGTGGAG AGTTTT 13988
GlyThrAspTrp LeuAlaAsn LysPheAsp TrpAlaValGlu SerPhe
55 60 65
GTCATCTTTCCC GTTTTGACT CACATTGTC TCCTATGGTGCC CTCACT 14036
ValIlePhePro ValLeuThr HisIleVal SerTyrGlyAla LeuThr
70 75 80
ACCAGCCATTTC CTTGACACA GTCGCTTTA GTCACTGTGTCT ACCGCC 14084
ThrSerHisPhe LeuAspThr ValAlaLeu ValThrValSer ThrAla
CA 02418780 2003-02-28
85 90 95
GGG TTT GTT CAC GGG CGG TAT GTC CTA AGT AGC ATC TAC GCG GTC TGT 14132
Gly Phe Val His Gly Arg Tyr Val Leu Ser Ser Ile Tyr Ala Val Cys
100 105 110 115
GCC CTG GCT GCG TTG ACT TGC TTC GTC ATT AGG TTT GCA AAG AAT TGC 14180
Ala Leu Ala Ala Leu Thr Cys Phe Val Ile Arg Phe Ala Lys Asn Cys
120 125 130
ATG TCC TGG CGC TAC GCG TGT ACC AGA TAT ACC AAC TTT CTT CTG GAC 14228
Met Ser Trp Arg Tyr Ala Cys Thr Arg Tyr Thr Asn Phe Leu Leu Asp
135 140 145
ACT AAG GGC AGA CTC TAT CGT TGG CGG TCG CCT GTC ATC ATA GAG AAA 14276
Thr Lys Gly Arg Leu Tyr Arg Trp Arg Ser Pro Val Ile Ile Glu Lys
150 155 160
AGG GGC AAA GTT GAG GTC GAA GGT CAT CTG ATC GAC CTC AAA AGA GTT 14324
Arg Gly Lys Val Glu Val Glu Gly His Leu Ile Asp Leu Lys Arg Val
165 170 175
GTG CTT GAT GGT TCC GTG GCA ACC CCT ATA ACC AGA GTT TCA GCG GAA 14372
Val Leu Asp Gly Ser Val Ala Thr Pro Ile Thr Arg Val Ser Ala Glu
180 185 190 195
CAA TGG GGT CGT CCT TAG ATGACTTCTG TCATGATAGC ACGGCTCCAC 14420
Gln Trp Gly Arg Pro
200
AAAAGGTGCT TTTGGCGTTT TCTATTACCT ACACGCCAGT GATGATATAT GCCCTAAAGG 14480
TGAGTCGCGG CCGACTGCTA GGGCTTCTGC ACCTTTTGAT CTTCCTGAAT TGTGCTTTCA 14540
CCTTCGGGTA CATGACTTTC GCGCACTTTC AGAGTACAAA TAAGGTCGCG CTCACTATGG 14600
GAGCAGTAGT TGCACTCCTT TGGGGGGTGT ACTCAGCCAT AGAAACCTGG AAATTCATCA 14660
CCTCCAGATG CCGTTTGTGC TTGCTAGGCC GCAAGTACAT TCTGGCCCCT GCCCACCACG 14720
TTGAAAGTGC CGCACGGTTT CATCCGATTG CGGCAAATGA TAACCACGCA TTTGTCGTCC 14780
GGCGTCCCGG CTCCACTACG GTCAACGGCA CATTGGTGCC CGGGTTAAAA AGCCTCGTGT 14840
TGGGTGGCAG AAAAGCTGTT AAACAGGGAG TGGTAAACCT TGTCAAATAT GCCAAATAAC 14900
AACGGCAAGC AGCAGAAGAG AAAGAAGGGG GATGGCCAGC CAGTCAATCA GCTGTGCCAG 14960
ATGCTGGGTA AGATCATCGC TCAGCAAAAC CAGTCCAGAG GCAAGGGACC GGGAAAGAAA 15020
AATAAGAAGA AAAACCCGGA GAAGCCCCAT TTTCCTCTAG CGACTGAAGA TGATGTCAGA 15080
CATCACTTTA CCCCTAGTGA GCGGCAATTG TGTCTGTCGT CAATCCAGAC CGCCTTTAAT 15140
CAAGGCGCTG GGACTTGCAC CCTGTCAGAT TCAGGGAGGA TAAGTTACAC TGTGGAGTTT 15200
AGTTTGCCTA CGCATCATAC TGTGCGCCTG ATCCGCGTCA CAGCATCACC CTCAGCATGA 15260
TGGGCTGGCA TTCTTGAGGC ATCTCAGTGT TTGAATTGGA AGAATGTGTG GTGAATGGCA 15320
CTGATTGACA TTGTGCCTCT AAGTCACCTA TTCAATTAGG GCGACCGTGT GGGGGTGAGA 15380
TTTAATTGGC GAGAACCATG CGGCCGAAAT T 15411
2) INFORMATION FOR SEQ ID N0:27:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH:200 amino acids
(B) TYPE: Amino Acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Protein
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Sus scrofa
31
CA 02418780 2003-02-28
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:27:
Met Leu Glu Lys Cys Leu Thr Ala Gly Cys Cys Ser Arg Leu Leu Ser
1 5 10 15
Leu Trp Cys Ile Val Pro Phe Cys Phe Ala Val Leu Ala Asn Ala Ser
20 25 30
Asn Asp Ser Ser Ser His Leu Gln Leu Ile Tyr Asn Leu Thr Leu Cys
35 40 45
Glu Leu Asn Gly Thr Asp Trp Leu Ala Asn Lys Phe Asp Trp Ala Val
50 55 60
Glu Ser Phe Val Ile Phe Pro Val Leu Thr His Ile Val Ser Tyr Gly
65 70 75 80
Ala Leu Thr Thr Ser His Phe Leu Asp Thr Val Ala Leu Val Thr Val
85 90 95
Ser Thr Ala Gly Phe Val His Gly Arg Tyr Val Leu Ser Ser Ile Tyr
100 105 110
Ala Val Cys Ala Leu Ala Ala Leu Thr Cys Phe Val Ile Arg Phe Ala
115 120 125
Lys Asn Cys Met Ser Trp Arg Tyr Ala Cys Thr Arg Tyr Thr Asn Phe
130 135 140
Leu Leu Asp Thr Lys Gly Arg Leu Tyr Arg Trp Arg Ser Pro Val Ile
145 150 155 160
Ile Glu Lys Arg Gly Lys Val Glu Val Glu Gly His Leu Ile Asp Leu
165 170 175
Lys Arg Val Val Leu Asp Gly Ser Val Ala Thr Pro Ile Thr Arg Val
180 185 190
Ser Ala Glu Gln Trp Gly Arg Pro
195 200
(2) INFORMATION FOR SEQ ID N0:28:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15411 base pairs
(B) TYPE: Nucleic acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(ii) MOLECULE TYPE: DNA
(vi) ORIGINAL SOURCE:
(A) ORGANISM:Sus scrofa
(ix) FEATURE:
(A) NAME/KEY:CDS
(B) LOCATION: (12073)...(12843)
(D) OTHER INFORMATION: wtORF2 of PRRSV strain VR-2332 GenBank
Accession #U87392
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:28:
ATGACGTATA GGTGTTGGCT CTATGCCTTG GCATTTGTAT TGTCAGGAGC TGTGACCATT 60
GGCACAGCCC AAAACTTGCT GCACAGAAAC ACCCTTCTGT GATAGCCTCC TTCAGGGGAG 120
CTTAGGGTTT GTCCCTAGCA CCTTGCTTCC GGAGTTGCAC TGCTTTACGG TCTCTCCACC 180
CCTTTAACCA TGTCTGGGAT ACTTGATCGG TGCACGTGTA CCCCCAATGC CAGGGTGTTT 240
ATGGCGGAGG GCCAAGTCTA CTGCACACGA TGCCTCAGTG CACGGTCTCT CCTTCCCCTG 300
AACCTCCAAG TTTCTGAGCT CGGGGTGCTA GGCCTATTCT ACAGGCCCGA AGAGCCACTC 360
CGGTGGACGT TGCCACGTGC ATTCCCCACT GTTGAGTGCT CCCCCGCCGG GGCCTGCTGG 420
CTTTCTGCAA TCTTTCCAAT CGCACGAATG ACCAGTGGAA ACCTGAACTT CCAACAAAGA 480
32
CA 02418780 2003-02-28
ATGGTACGGG TCGCAGCTGA GCTTTACAGA GCCGGCCAGC TCACCCCTGC AGTCTTGAAG 540
GCTCTACAAG TTTATGAACG GGGTTGCCGC TGGTACCCCA TTGTTGGACC TGTCCCTGGA 600
GTGGCCGTTT TCGCCAATTC CCTACATGTG AGTGATAAAC CTTTCCCGGG AGCAACTCAC 660
GTGTTGACCA ACCTGCCGCT CCCGCAGAGA CCCAAGCCTG AAGACTTTTG CCCCTTTGAG 720
TGTGCTATGG CTACTGTCTA TGACATTGGT CATGACGCCG TCATGTATGT GGCCGAAAGG 780
AAAGTCTCCT GGGCCCCTCG TGGCGGGGAT GAAGTGAAAT TTGAAGCTGT CCCCGGGGAG 840
TTGAAGTTGA TTGCGAACCG GCTCCGCACC TCCTTCCCGC CCCACCACAC AGTGGACATG 900
TCTAAGTTCG CCTTCACAGC CCCTGGGTGT GGTGTTTCTA TGCGGGTCGA ACGCCAACAC 960
GGCTGCCTTC CCGCTGACAC TGTCCCTGAA GGCAACTGCT GGTGGAGCTT GTTTGACTTG 1020
CTTCCACTGG AAGTTCAGAA CAAAGAAATT CGCCATGCTA ACCAATTTGG CTACCAGACC 1080
AAGCATGGTG TCTCTGGCAA GTACCTACAG CGGAGGCTGC AAGTTAATGG TCTCCGAGCA 1140
GTAACTGACC TAAACGGACC TATCGTCGTA CAGTACTTCT CCGTTAAGGA GAGTTGGATC 1200
CGCCATTTGA AACTGGCGGG AGAACCCAGC TACTCTGGGT TTGAGGACCT CCTCAGAATA 1260
AGGGTTGAGC CTAACACGTC GCCATTGGCT GACAAGGAAG AAAAAATTTT CCGGTTTGGC 1320
AGTCACAAGT GGTACGGCGC TGGAAAGAGA GCAAGAAAAG CACGCTCTTG TGCGACTGCT 1380
ACAGTCGCTG GCCGCGCTTT GTCCGTTCGT GAAACCCGGC AGGCCAAGGA GCACGAGGTT 1440
GCCGGCGCCA ACAAGGCTGA GCACCTCAAA CACTACTCCC CGCCTGCCGA AGGGAATTGT 1500
GGTTGGCACT GCATTTCCGC CATCGCCAAC CGGATGGTGA ATTCCAAATT TGAAACCACC 1560
CTTCCCGAAA GAGTGAGACC TCCAGATGAC TGGGCTACTG ACGAGGATCT TGTGAATGCC 1620
ATCCAAATCC TCAGACTCCC TGCGGCCTTA GACAGGAACG GTGCTTGTAC TAGCGCCAAG 1680
TACGTACTTA AGCTGGAAGG TGAGCATTGG ACTGTCACTG TGACCCCTGG GATGTCCCCT 1740
TCTTTGCTCC CTCTTGAATG TGTTCAGGGC TGTTGTGGGC ACAAGGGCGG TCTTGGTTCC 1800
CCAGATGCAG TCGAGGTCTC CGGATTTGAC CCTGCCTGCC TTGACCGGCT GGCTGAGGTG 1860
ATGCACCTGC CTAGCAGTGC TATCCCAGCC GCTCTGGCCG AAATGTCTGG CGATTCCGAT 1920
CGTTCGGCTT CTCCGGTCAC CACCGTGTGG ACTGTTTCGC AGTTCTTTGC CCGTCACAGC 1980
GGAGGGAATC ACCCTGACCA AGTGCGCTTA GGGAAAATTA TCAGCCTTTG TCAGGTGATT 2040
GAGGACTGCT GCTGTTCCCA GAACAAAACC AACCGGGTCA CCCCGGAGGA GGTCGCAGCA 2100
AAGATTGACC TGTACCTCCG TGGTGCAACA AATCTTGAAG AATGCTTGGC CAGGCTTGAG 2160
AAAGCGCGCC CGCCACGCGT AATCGACACC TCCTTTGATT GGGATGTTGT GCTCCCTGGG 2220
GTTGAGGCGG CAACCCAGAC GATCAAGCTG CCCCAGGTCA ACCAGTGTCG TGCTCTGGTC 2280
CCTGTTGTGA CTCAAAAGTC CTTGGACAAC AACTCGGTCC CCCTGACCGC CTTTTCACTG 2340
GCTAACTACT ACTACCGTGC GCAAGGTGAC GAAGTTCGTC ACCGTGAAAG ACTAACCGCC 2400
GTGCTCTCCA AGTTGGAAAA GGTTGTTCGA GAAGAATATG GGCTCATGCC AACCGAGCCT 2460
GGTCCACGGC CCACACTGCC ACGCGGGCTC GACGAACTCA AAGACCAGAT GGAGGAGGAC 2520
TTGCTGAAAC TGGCTAACGC CCAGACGACT TCGGACATGA TGGCCTGGGC AGTCGAGCAG 2580
GTTGACCTAA AAACTTGGGT CAAGAACTAC CCGCGGTGGA CACCACCACC CCCTCCGCCA 2640
AAAGTTCAGC CTCGAAAAAC GAAGCCTGTC AAGAGCTTGC CGGAGAGAAA GCCTGTCCCC 2700
GCCCCGCGCA GGAAGGTTGG GTCCGATTGT GGCAGCCCGG TTTCATTAGG CGGCGATGTC 2760
CCTAACAGTT GGGAAGATTT GGCTGTTAGT AGCCCCTTTG ATCTCCCGAC CCCACCTGAG 2820
CCGGCAACAC CTTCAAGTGA GCTGGTGATT GTGTCCTCAC CGCAATGCAT CTTCAGGCCG 2880
GCGACACCCT TGAGTGAGCC GGCTCCAATT CCCGCACCTC GCGGAACTGT GTCTCGACCG 2940
GTGACACCCT TGAGTGAGCC GATCCCTGTG CCCGCACCGC GGCGTAAGTT TCAGCAGGTG 3000
AAAAGATTGA GTTCGGCGGC GGCAATCCCA CCGTACCAGG ACGAGCCCCT GGATTTGTCT 3060
GCTTCCTCAC AGACTGAATA TGAGGCCTCT CCCCCAGCAC CGCCGCAGAG CGGGGGCGTT 3120
CTGGGAGTAG AGGGGCATGA AGCTGAGGAA ACCCTGAGTG AAATCTCGGA CATGTCGGGT 3180
AACATTAAAC CTGCGTCCGT GTCATCAAGC AGCTCCTTGT CCAGCGTGAG AATCACACGC 3240
CCAAAATACT CAGCTCAAGC CATCATCGAC TCGGGCGGGC CCTGCAGTGG GCATCTCCAA 3300
GAGGTAAAGG AAACATGCCT TAGTGTCATG CGCGAGGCAT GTGATGCGAC TAAGCTTGAT 3360
GACCCTGCTA CGCAGGAATG GCTTTCTCGC ATGTGGGATC GGGTGGACAT GCTGACTTGG 3420
CGCAACACGT CTGTTTACCA GGCGATTTGC ACCTTAGATG GCAGGTTAAA GTTCCTCCCA 3480
AAAATGATAC TCGAGACACC GCCGCCCTAT CCGTGTGAGT TTGTGATGAT GCCTCACACG 3540
CCTGCACCTT CCGTAGGTGC GGAGAGCGAC CTTACCATTG GCTCAGTTGC TACTGAAGAT 3600
GTTCCACGCA TCCTCGAGAA AATAGAAAAT GTCGGCGAGA TGGCCAACCA GGGACCCTTG 3660
GCCTTCTCCG AGGATAAACC GGTAGATGAC CAACTTGTCA ACGACCCCCG GATATCGTCG 3720
CGGAGGCCTG ACGAGAGCAC ATCAGCTCCG TCCGCAGGCA CAGGTGGCGC CGGCTCTTTT 3780
ACCGATTTGC CGCCTTCAGA TGGCGCGGAT GCGGACGGGG GGGGGCCGTT TCGGACGGTA 3840
AAAAGAAAAG CTGAAAGGCT CTTTGACCAA CTGAGCCGTC AGGTTTTTGA CCTCGTCTCC 3900
CATCTCCCTG TTTTCTTCTC ACGCCTTTTC TACCCTGGCG GTGGTTATTC TCCGGGTGAT 3960
TGGGGTTTTG CAGCTTTTAC TCTATTGTGC CTCTTTTTAT GTTACAGTTA CCCAGCCTTT 4020
GGTATTGCTC CCCTCTTGGG TGTGTTTTCT GGGTCTTCTC GGCGCGTTCG AATGGGGGTT 4080
TTTGGCTGCT GGTTGGCTTT TGCTGTTGGT CTGTTCAAGC CTGTGTCCGA CCCAGTCGGC 4140
33
CA 02418780 2003-02-28
GCTGCTTGTG AGTTTGACTC GCCAGAGTGT AGAAACATCC TTCATTCTTT TGAGCTTCTC 4200
AAACCTTGGG ACCCTGTTCG CAGCCTTGTT GTGGGCCCCG TCGGTCTCGG TCTTGCCATT 4260
CTTGGCAGGT TACTGGGCGG GGCACGCTGC ATCTGGCACT TTTTGCTTAG GCTTGGCATT 4320
GTTGCAGACT GTATCTTGGC TGGAGCTTAC GTGCTTTCTC AAGGTAGGTG TAAAAAGTGC 4380
TGGGGATCTT GTATAAGAAC TGCTCCTAAT GAGGTCGCTT TTAACGTGTT TCCTTTCACA 4440
CGTGCGACCA GGTCGTCACT TATCGACCTG TGCGATCGGT TTTGTGCGCC AAAAGGAATG 4500
GACCCCATTT TTCTCGCCAC TGGGTGGCGC GGGTGCTGGG CCGGCCGAAG CCCCATTGAG 4560
CAACCCTCTG AAAAACCCAT CGCGTTTGCC CAATTGGATG AAAAGAAGAT TACGGCTAGG 4620
ACTGTGGTCG CCCAGCCTTA TGACCCCAAC CAAGCCGTAA AGTGCTTGCG GGTATTGCAG 4680
TCGGGTGGGG CGATGGTGGC TAAGGCGGTC CCAAAAGTGG TCAAGGTTTC CGCTGTTCCA 4740
TTCCGAGCCC CCTTCTTTCC CACTGGAGTG AAAGTTGACC CTGATTGCAG GGTCGTGGTT 4800
GACCCTGACA CTTTCACTGC AGCTCTCCGG TCTGGCTACT CCACCACAAA CCTCGTCCTT 4860
GGTGTAGGGG ACTTTGCCCA GCTGAATGGA TTAAAAATCA GGCAAATTTC CAAGCCTTCA 4920
GGGGGAGGCC CACATCTCAT GGCTGCCCTG CATGTTGCCT GCTCGATGGC TCTGCACATG 4980
CTTGCTGGGA TTTATGTGAC TGCGGTGGGT TCTTGCGGCA CCGGCACCAA CGACCCGTGG 5040
TGCGCTAACC CGTTTGCCGT CCCTGGCTAC GGACCTGGCT CTCTCTGCAC GTCCAGGTTG 5100
TGCATTTCCC AACACGGCCT TACCCTGCCC TTGACAGCAC TTGTGGCGGG ATTCGGTATT 5160
CAAGAAATTG CCTTGGTCGT TTTGATTTTT GTTTCCATCG GAGGCATGGC TCATAGGTTG 5220
AGCTGTAAGG CTGACATGCT GTGTGTTTTG CTTGCAATTG CCAGCTATGT TTGGGTACCT 5280
CTTACCTGGT TGCTTTGTGT GTTTCCTTGC TGGTTGCGCT GTTTTTCTTT GCACCCCCTC 5340
ACCATCCTAT GGTTGGTGTT TTTCTTGATT TCTGTGAATA TGCCTTCAGG AATCTTGGCC 5400
ATGGTGTTGT TGGTTTCTCT TTGGCTTCTT GGTCGTTATA CTAATGTTGC TGGCCTTGTC 5460
ACCCCCTACG ACATTCATCA TTACACCAGT GGCCCCCGCG GTGTTGCCGC CTTGGCTACC 5520
GCACCAGATG GGACCTACTT GGCCGCTGTC CGCCGCGCTG CGTTGACTGG CCGCACCATG 5580
CTGTTTACCC CGTCCCAGCT TGGGTCTCTT CTTGAGGGTG CTTTCAGAAC TCGAAAGCCC 5640
TCACTGAACA CCGTCAATGT GATCGGGTCC TCCATGGGCT CTGGCGGGGT GTTTACCATC 5700
GACGGGAAAG TCAAGTGCGT AACTGCCGCA CATGTCCTTA CGGGCAATTC AGCTCGGGTT 5760
TCCGGGGTCG GCTTCAATCA AATGCTTGAC TTTGACGTAA AGGGAGATTT CGCTATAGCT 5820
GATTGCCCGA ATTGGCAAGG GGCTGCCCCC AAGACCCAAT TCTGCACGGA TGGATGGACT 5880
GGCCGTGCCT ATTGGCTAAC ATCCTCTGGC GTCGAACCCG GCGTCATTGG AAAAGGATTC 5940
GCCTTCTGCT TCACCGCATG TGGCGATTCC GGGTCCCCAG TGATCACCGA GGCCGGTGAG 6000
CTTGTCGGCG TTCACACGGG ATCGAATAAA CAAGGGGGGG GCATTGTTAC GCGCCCCTCA 6060
GGCCAGTTTT GTAATGTGGC ACCCATCAAG CTAAGCGAAT TAAGTGAATT CTTTGCTGGG 6120
CCTAAGGTCC CGCTCGGTGA TGTGAAGGTC GGCAGCCACA TAATTAAAGA CATAAGCGAG 6180
GTGCCTTCAG ATCTTTGTGC CTTGCTTGCT GCCAAACCTG AACTGGAAGG AGGCCTCTCC 6240
ACCGTCCAAC TTCTTTGTGT GTTTTTTCTC CTGTGGAGAA TGATGGGACA TGCCTGGACG 6300
CCCTTGGTTG CTGTGAGTTT CTTTATTTTG AATGAGGTTC TCCCAGCCGT CCTGGTCCGG 6360
AGTGTTTTCT CCTTTGGAAT GTTTGTGCTA TCCTGGCTCA CGCCATGGTC TGCGCAAGTT 6420
CTGATGATCA GGCTTCTGAC AGCAGCTCTT AACAGGAACA GATGGTCACT TGCCTTTTTC 6480
AGCCTCGGTG CAGTGACCGG TTTTGTCGCA GATCTTGCGG CCACTCAGGG GCATCCGTTG 6540
CAGGCAGTGA TGAATTTGAG CACCTATGCA TTCCTGCCTC GGATGATGGT TGTGACCTCA 6600
CCAGTCCCAG TGATCACGTG TGGTGTCGTG CACCTACTTG CCATCATTTT GTACTTGTTT 6660
AAGTACCGTG GCCCGCACCA TATCCTTGTT GGCGATGGAG TGTTCTCTGC GGCTTTCTTC 6720
TTGAGATACT TTGCCGAGGG AAAGTTGAGG GAAGGGGTGT CGCAATCCTG CGGAATGAAT 6780
CATGAGTCTC TGACTGGTGC CCTCGCTATG AGACTCAATG ACGAGGACTT GGATTTCCTT 6840
ATGAAATGGA CTGATTTTAA GTGCTTTGTT TCTGCGTCCA ACATGAGGAA TGCAGCGGGT 6900
CAATTTATCG AGGCTGCCTA TGCTAAAGCA CTTAGAGTAG AACTGGCCCA GTTGGTGCAG 6960
GTTGATAAAG TTCGAGGTAC TTTGGCCAAA CTTGAAGCTT TTGCTGATAC CGTGGCACCT 7020
CAACTCTCGC CCGGTGACAT TGTTGTCGCT CTCGGCCACA CGCCTGTTGG CAGTATCTTC 7080
GACCTAAAGG TTGGTAGCAC CAAGCATACC CTCCAAGCCA TTGAGACCAG AGTCCTTGCT 7140
GGGTCCAAAA TGACCGTGGC GCGCGTCGTC GACCCGACCC CCACGCCCCC ACCCGCACCC 7200
GTGCCCATCC CCCTCCCACC GAAAGTTCTG GAGAATGGCC CCAACGCTTG GGGGGATGAG 7260
GACCGTTTGA ATAAGAAGAA GAGGCGCAGG ATGGAAGCCC TCGGCATCTA TGTTATGGGC 7320
GGGAAAAAGT ACCAGAAATT TTGGGACAAG AATTCCGGTG ATGTGTTTTA TGAGGAGGTC 7380
CATAATAACA CAGATGAGTG GGAGTGTCTC AGAGTTGGCG ACCCTGCCGA CTTTGACCCT 7440
GAGAAGGGAA CTCTGTGTGG ACATGTCACC ATTGAAAACA AGGCTTACCA TGTTTACACC 7500
TCCCCATCTG GTAAGAAGTT CTTGGTCCCC GTCAACCCAG AGAATGGAAG AGTTCAATGG 7560
GAAGCTGCAA AGCTTTCCGT GGAGCAGGCC CTAGGTATGA TGAATGTCGA CGGCGAACTG 7620
ACTGCCAAAG AACTGGAGAA ACTGAAAAGA ATAATTGACA AACTCCAGGG CCTGACTAAG 7680
GAGCAGTGTT TAAACTGCTA GCCGCCAGCG ACTTGACCCG CTGTGGTCGC GGCGGCTTGG 7740
TTGTTACTGA AACAGCGGTA AAAATAGTCA AATTTCACAA CCGGACCTTC ACCCTGGGAC 7800
CTGTGAATTT AAAAGTGGCC AGTGAGGTTG AGCTAAAAGA CGCGGTTGAG CACAACCAAC 7860
34
CA 02418780 2003-02-28
ACCCGGTTGC GAGACCGATC GATGGTGGAG TTGTGCTCCT GCGTTCCGCG GTTCCTTCGC 7920
TTATAGACGT CTTGATCTCC GGTGCTGATG CATCTCCCAA GTTACTTGCC CATCACGGGC 7980
CGGGAAACAC TGGGATCGAT GGCACGCTCT GGGATTTTGA GTCCGAAGCC ACTAAAGAGG 8040
AAGTCGCACT CAGTGCGCAA ATAATACAGG CTTGTGACAT TAGGCGCGGC GACGCTCCTG 8100
AAATTGGTCT CCCTTACAAG CTGTACCCTG TTAGGGGTAA CCCTGAGCGG GTGAAAGGAG 8160
TTCTGCAGAA TACAAGGTTT GGAGACATAC CTTACAAAAC CCCCAGTGAC ACTGGAAGCC 8220
CAGTGCACGC GGCTGCCTGC CTTACGCCCA ACGCCACTCC GGTGACTGAT GGGCGCTCCG 8280
TCTTGGCCAC GACCATGCCC CCCGGGTTTG AGTTATATGT ACCGACCATA CCAGCGTCTG 8340
TCCTTGATTA CCTTGACTCT AGGCCTGACT GCCCTAAACA GCTGACAGAG CACGGCTGCG 8400
AAGATGCCGC ACTGAAAGAC CTCTCTAAAT ATGACTTGTC CACCCAAGGC TTTGTTTTAC 8460
CTGGAGTTCT TCGCCTTGTG CGGAAATACC TGTTTGCCCA TGTAGGTAAG TGCCCACCCG 8520
TTCATCGGCC TTCTACTTAC CCTGCTAAGA ATTCTATGGC TGGAATAAAT GGGAACAGGT 8580
TCCCAACCAA GGACATTCAG AGCGTCCCTG AAATCGACGT TCTGTGCGCA CAGGCTGTGC 8640
GAGAAAACTG GCAAACTGTC ACCCCTTGTA CTCTTAAGAA ACAGTATTGC GGGAAGAAGA 8700
AGACTAGGAC CATACTCGGC ACCAATAACT TCATCGCACT AGCCCACCGA GCAGTGTTGA 8760
GTGGTGTTAC CCAGGGCTTC ATGAAAAAGG CGTTTAACTC GCCCATCGCC CTCGGAAAGA 8820
ACAAGTTTAA GGAGCTACAG ACTCCGGTCC TGGGCAGGTG CCTTGAAGCT GATCTCGCAT 8880
CCTGCGATCG ATCCACGCCT GCAATTGTCC GCTGGTTTGC CGCCAACCTT CTTTATGAAC 8940
TTGCCTGTGC TGAAGAGCAT CTACCGTCGT ACGTGCTGAA CTGCTGCCAC GACTTACTGG 9000
TCACGCAGTC CGGCGCAGTG ACTAAGAGAG GTGGCCTGTC GTCTGGCGAC CCGATCACCT 9060
CTGTGTCTAA CACCATTTAT AGTTTGGTGA TCTATGCACA GCATATGGTG CTTAGTTACT 9120
TCAAAAGTGG TCACCCCCAT GGCCTTCTGT TCTTACAAGA CCAGCTAAAG TTTGAGGACA 9180
TGCTCAAGGT TCAACCCCTG ATCGTCTATT CGGACGACCT CGTGCTGTAT GCCGAGTCTC 9240
CCACCATGCC AAACTATCAC TGGTGGGTTG AACATCTGAA TTTGATGCTG GGGTTTCAGA 9300
CGGACCCAAA GAAGACAGCA ATAACAGACT CGCCATCATT TCTAGGCTGT AGAATAATAA 9360
ATGGGCGCCA GCTAGTCCCC AACCGTGACA GGATCCTCGC GGCCCTCGCC TATCACATGA 9420
AGGCGAGTAA TGTTTCTGAA TACTATGCCT CAGCGGCTGC AATACTCATG GACAGCTGTG 9480
CTTGTTTGGA GTATGATCCT GAATGGTTTG AAGAACTTGT AGTTGGAATA GCGCAGTGCG 9540
CCCGCAAGGA CGGCTACAGC TTTCCCGGCA CGCCGTTCTT CATGTCCATG TGGGAAAAAC 9600
TCAGGTCCAA TTATGAGGGG AAGAAGTCGA GAGTGTGCGG GTACTGCGGG GCCCCGGCCC 9660
CGTACGCTAC TGCCTGTGGC CTCGACGTCT GCATTTACCA CACCCACTTC CACCAGCATT 9720
GTCCAGTCAC AATCTGGTGT GGCCATCCAG CGGGTTCTGG TTCTTGTAGT GAGTGCAAAT 9780
CCCCTGTAGG GAAAGGCACA AGCCCTTTAG ACGAGGTGCT GGAACAAGTC CCGTATAAGC 9840
CCCCACGGAC CGTTATCATG CATGTGGAGC AGGGTCTCAC CCCCCTTGAT CCAGGTAGAT 9900
ACCAAACTCG CCGCGGATTA GTCTCTGTCA GGCGTGGAAT TAGGGGAAAT GAAGTTGGAC 9960
TACCAGACGG TGATTATGCT AGCACCGCCT TGCTCCCTAC CTGCAAAGAG ATCAACATGG 10020
TCGCTGTCGC TTCCAATGTA TTGCGCAGCA GGTTCATCAT CGGCCCACCC GGTGCTGGGA 10080
AAACATACTG GCTCCTTCAA CAGGTCCAGG ATGGTGATGT TATTTACACA CCAACTCACC 10140
AGACCATGCT TGACATGATT AGGGCTTTGG GGACGTGCCG GTTCAACGTC CCGGCAGGCA 10200
CAACGCTGCA ATTCCCCGTC CCCTCCCGCA CCGGTCCGTG GGTTCGCATC CTAGCCGGCG 10260
GTTGGTGTCC TGGCAAGAAT TCCTTCCTAG ATGAAGCAGC GTATTGCAAT CACCTTGATG 10320
TTTTGAGGCT TCTTAGTAAA ACTACCCTCA CCTGTCTAGG AGACTTCAAG CAACTCCACC 10380
CAGTGGGTTT TGATTCTCAT TGCTATGTTT TTGACATCAT GCCTCAAACT CAACTGAAGA 10440
CCATCTGGAG GTTTGGACAG AATATCTGTG ATGCCATTCA GCCAGATTAC AGGGACAAAC 10500
TCATGTCCAT GGTCAACACA ACCCGTGTGA CCTACGTGGA AAAACCTGTC AGGTATGGGC 10560
AGGTCCTCAC CCCCTACCAC AGGGACCGAG AGGACGACGC CATCACTATT GACTCCAGTC 10620
AAGGCGCCAC ATTCGATGTG GTTACATTGC ATTTGCCCAC TAAAGATTCA CTCAACAGGC 10680
AAAGAGCCCT TGTTGCTATC ACCAGGGCAA GACACGCTAT CTTTGTGTAT GACCCACACA 10740
GGCAGCTGCA GGGCTTGTTT GATCTTCCTG CAAAAGGCAC GCCCGTCAAC CTCGCAGTGC 10800
ACTGCGACGG GCAGCTGATC GTGCTGGATA GAAATAACAA AGAATGCACG GTTGCTCAGG 10860
CTCTAGGCAA CGGGGATAAA TTTAGGGCCA CAGACAAGCG TGTTGTAGAT TCTCTCCGCG 10920
CCATTTGTGC TGATCTAGAA GGGTCGAGCT CTCCGCTCCC CAAGGTCGCA CACAACTTGG 10980
GATTTTATTT CTCACCTGAT TTAACACAGT TTGCTAAACT CCCAGTAGAA CTTGCACCTC 11040
ACTGGCCCGT GGTGTCAACC CAGAACAATG AAAAGTGGCC GGATCGGCTG GTTGCCAGCC 11100
TTCGCCCTAT CCATAAATAC AGCCGCGCGT GCATCGGTGC CGGCTATATG GTGGGCCCTT 11160
CGGTGTTTCT AGGCACTCCT GGGGTCGTGT CATACTATCT CACAAAATTT GTTAAGGGCG 11220
GGGCTCAAGT GCTTCCGGAG ACGGTTTTCA GCACCGGCCG AATTGAGGTA GACTGCCGGG 11280
AATATCTTGA TGATCGGGAG CGAGAAGTTG CTGCGTCCCT CCCACACGGT TTCATTGGCG 11340
ACGTCAAAGG CACTACCGTT GGAGGATGTC ATCATGTCAC CTCCAGATAC CTCCCGCGCG 11400
TCCTTCCCAA GGAATCAGTT GCGGTAGTCG GGGTTTCAAG CCCCGGAAAA GCCGCGAAAG 11460
CATTGTGCAC ACTGACAGAT GTGTACCTCC CAGATCTTGA AGCCTATCTC CACCCGGAGA 11520
CCCAGTCCAA GTGCTGGAAA ATGATGTTGG ACTTCAAAGA AGTTCGACTA ATGGTCTGGA 11580
CA 02418780 2003-02-28
AAGACAAAAC AGCCTATTTC CAACTTGAAG GTCGCTATTT CACCTGGTAT CAGCTTGCCA 11640
GCTATGCCTC GTACATCCGT GTTCCCGTCA ACTCTACGGT GTACTTGGAC CCCTGCATGG 11700
GCCCCGCCCT TTGCAACAGG AGAGTCGTCG GGTCCACCCA CTGGGGGGCT GACCTCGCGG 11760
TCACCCCTTA TGATTACGGC GCTAAAATTA TCCTGTCTAG CGCGTACCAT GGTGAAATGC 11820
CCCCCGGATA CAAAATTCTG GCGTGCGCGG AGTTCTCGTT GGATGACCCA GTTAAGTACA 11880
AACATACCTG GGGGTTTGAA TCGGATACAG CGTATCTGTA TGAGTTCACC GGAAACGGTG 11940
AGGACTGGGA GGATTACAAT GATGCGTTTC GTGCGCGCCA GGAAGGGAAA ATTTATAAGG 12000
CCACTGCCAC CAGCTTGAAG TTTTATTTTC CCCCGGGCCC TGTCATTGAA CCAACTTTAG 12060
GCCTGAATTG AA ATG AAA TGG GGT CCA TGC AAA GCC TTT TTG ACA AAA TTG 12111
Met Lys Trp Gly Pro Cys Lys Ala Phe Leu Thr Lys Leu
1 5 10
GCC AAC TTT TTG TGG ATG CTT TCA CGG AGT TCT TGG TGT CCA TTG TTG 12159
Ala Asn Phe Leu Trp Met Leu Ser Arg Ser Ser Trp Cys Pro Leu Leu
15 20 25
ATA TCA TTA TAT TTT TGG CCA TTT TGT TTG GCT TCA CCA TCG CCG GTT 12207
Ile Ser Leu Tyr Phe Trp Pro Phe Cys Leu Ala Ser Pro Ser Pro Val
30 35 40 45
GGC TGG TGG TCT TTT GCA TCA GAT TGG TTT GCT CCG CGA TAC TCC GTA 12255
Gly Trp Trp Ser Phe Ala Ser Asp Trp Phe Ala Pro Arg Tyr Ser Val
50 55 60
CGC GCC CTG CCA TTC ACT CTG AGC AAT TAC AGA AGA TCT TAT GAG GCC 12303
Arg Ala Leu Pro Phe Thr Leu Ser Asn Tyr Arg Arg Ser Tyr Glu Ala
65 70 75
TTT CTT TCC CAG TGC CAA GTG GAC ATT CCC ACC TGG GGA ACT AAA CAT 12351
Phe Leu Ser Gln Cys Gln Val Asp Ile Pro Thr Trp Gly Thr Lys His
BO 85 90
CCT TTG GGG ATG CTT TGG CAC CAT AAG GTG TCA ACC CTG ATT GAT GAA 12399
Pro Leu Gly Met Leu Trp His His Lys Val Ser Thr Leu Ile Asp Glu
95 100 105
ATG GTG TCG CGT CGA ATG TAC CGC ATC ATG GAA AAA GCA GGG CAG GCT 12447
Met Val Ser Arg Arg Met Tyr Arg Ile Met Glu Lys Ala Gly Gln Ala
110 115 120 125
GCC TGG AAA CAG GTG GTG AGC GAG GCT ACG CTG TCT CGC ATT AGT AGT 12495
Ala Trp Lys Gln Val Val Ser Glu Ala Thr Leu Ser Arg Ile Ser Ser
130 135 140
TTG GAT GTG GTG GCT CAT TTT CAG CAT CTA GCC GCC ATT GAA GCC GAG 12543
Leu Asp Val Val Ala His Phe Gln His Leu Ala Ala Ile Glu Ala Glu
145 150 155
ACC TGT AAA TAT TTG GCC TCC CGG CTG CCC ATG CTA CAC AAC CTG CGC 12591
Thr Cys Lys Tyr Leu Ala Ser Arg Leu Pro Met Leu His Asn Leu Arg
160 165 170
ATG ACA GGG TCA AAT GTA ACC ATA GTG TAT AAT AGC ACT TTG AAT CAG 12639
Met Thr Gly Ser Asn Val Thr Ile Val Tyr Asn Ser Thr Leu Asn Gln
175 180 185
GTG TTT GCT ATT TTT CCA ACC CCT GGT TCC CGG CCA AAG CTT CAT GAT 12687
Val Phe Ala Ile Phe Pro Thr Pro Gly Ser Arg Pro Lys Leu His Asp
190 195 200 205
TTT CAG CAA TGG TTA ATA GCT GTA CAT TCC TCC ATA TTT TCC TCT GTT 12735
Phe Gln Gln Trp Leu Ile Ala Val His Ser Ser Ile Phe Ser Ser Val
36
CA 02418780 2003-02-28
210 215 220
GCA GCT TCT TGT ACT CTT TTT GTT GTG CTG TGG TTG CGG GTT CCA ATA 12783
Ala Ala Ser Cys Thr Leu Phe Val Val Leu Trp Leu Arg Val Pro Ile
225 230 235
CTA CGT ACT GTT TTT GGT TTC CGC TGG TTA GGG GCA ATT TTT CTT TCG 12831
Leu Arg Thr Val Phe Gly Phe Arg Trp Leu Gly Ala Ile Phe Leu Ser
240 245 250
AAC TCA CAG TGA ATTACACGGT GTGTCCACCT TGCCTCACCC GGCAAGCAGC 12883
Asn Ser Gln
255
CACAGAGATC TACGAACCCG GTAGGTCTCT TTGGTGCAGG ATAGGGTATG ACCGATGTGG 12943
GGAGGACGAT CATGACGAGC TAGGGTTTAT GATACCGCCT GGCCTCTCCA GCGAAGGCCA 13003
CTTGACTGGT GTTTACGCCT GGTTGGCGTT CTTGTCCTTC AGCTACACGG CCCAGTTCCA 13063
TCCCGAGATA TTCGGGATAG GGAATGTGAG TCGAGTTTAT GTTGACATCA AACATCAACT 13123
CATCTGCGCC GAACATGACG GGCAGAACAC CACCTTGCCT CGTCATGACA ACATTTCAGC 13183
CGTGTTTCAG ACCTATTACC AACATCAAGT CGACGGCGGC AATTGGTTTC ACCTAGAATG 13243
GCTTCGTCCC TTCTTTTCCT CGTGGTTGGT TTTAAATGTC TCTTGGTTTC TCAGGCGTTC 13303
GCCTGCAAAC CATGTTTCAG TTCGAGTCTT GCAGATATTA AGACCAACAC CACCGCAGCG 13363
GCAAGCTTTG CTGTCCTCCA AGACATCAGT TGCCTTAGGC ATCGCGACTC GGCCTCTGAG 13423
GCGATTCGCA AAATCCCTCA GTGCCGTACG GCGATAGGGA CACCCGTGTA TGTTACCATC 13483
ACAGCCAATG TGACAGATGA GAATTATTTA CATTCTTCTG ATCTCCTCAT GCTTTCTTCT 13543
TGCCTTTTCT ATGCTTCTGA GATGAGTGAA AAGGGATTTA AGGTGGTATT TGGCAATGTG 13603
TCAGGCATCG TGGCTGTGTG TGTCAATTTT ACCAGCTACG TCCAACATGT CAAGGAGTTT 13663
ACCCAACGCT CCCTGGTGGT CGACCATGTG CGGTTGCTCC ATTTCATGAC ACCTGAGACC 13723
ATGAGGTGGG CAACTGTTTT AGCCTGTCTT TTTGCCATTC TGTTGGCAAT TTGAATGTTT 13783
AAGTATGTTG GAGAAATGCT TGACCGCGGG CTGTTGCTCG CGATTGCTTT CTTTGTGGTG 13843
TATCGTGCCG TTCTGTTTTG CTGTGCTCGC CAACGCCAGC AACGACAGCA GCTCCCATCT 13903
ACAGCTGATT TACAACTTGA CGCTATGTGA GCTGAATGGC ACAGATTGGC TAGCTAACAA 13963
ATTTGATTGG GCAGTGGAGA GTTTTGTCAT CTTTCCCGTT TTGACTCACA TTGTCTCCTA 14023
TGGTGCCCTC ACTACCAGCC ATTTCCTTGA CACAGTCGCT TTAGTCACTG TGTCTACCGC 14083
CGGGTTTGTT CACGGGCGGT ATGTCCTAAG TAGCATCTAC GCGGTCTGTG CCCTGGCTGC 14143
GTTGACTTGC TTCGTCATTA GGTTTGCAAA GAATTGCATG TCCTGGCGCT ACGCGTGTAC 14203
CAGATATACC AACTTTCTTC TGGACACTAA GGGCAGACTC TATCGTTGGC GGTCGCCTGT 14263
CATCATAGAG AAAAGGGGCA AAGTTGAGGT CGAAGGTCAT CTGATCGACC TCAAAAGAGT 14323
TGTGCTTGAT GGTTCCGTGG CAACCCCTAT AACCAGAGTT TCAGCGGAAC AATGGGGTCG 14383
TCCTTAGATG ACTTCTGTCA TGATAGCACG GCTCCACAAA AGGTGCTTTT GGCGTTTTCT 14443
ATTACCTACA CGCCAGTGAT GATATATGCC CTAAAGGTGA GTCGCGGCCG ACTGCTAGGG 14503
CTTCTGCACC TTTTGATCTT CCTGAATTGT GCTTTCACCT TCGGGTACAT GACTTTCGCG 14563
CACTTTCAGA GTACAAATAA GGTCGCGCTC ACTATGGGAG CAGTAGTTGC ACTCCTTTGG 14623
GGGGTGTACT CAGCCATAGA AACCTGGAAA TTCATCACCT CCAGATGCCG TTTGTGCTTG 14683
CTAGGCCGCA AGTACATTCT GGCCCCTGCC CACCACGTTG AAAGTGCCGC ACGGTTTCAT 14743
CCGATTGCGG CAAATGATAA CCACGCATTT GTCGTCCGGC GTCCCGGCTC CACTACGGTC 14803
AACGGCACAT TGGTGCCCGG GTTAAAAAGC CTCGTGTTGG GTGGCAGAAA AGCTGTTAAA 14863
CAGGGAGTGG TAAACCTTGT CAAATATGCC AAATAACAAC GGCAAGCAGC AGAAGAGAAA 14923
GAAGGGGGAT GGCCAGCCAG TCAATCAGCT GTGCCAGATG CTGGGTAAGA TCATCGCTCA 14983
GCAAAACCAG TCCAGAGGCA AGGGACCGGG AAAGAAAAAT AAGAAGAAAA ACCCGGAGAA 15043
GCCCCATTTT CCTCTAGCGA CTGAAGATGA TGTCAGACAT CACTTTACCC CTAGTGAGCG 15103
GCAATTGTGT CTGTCGTCAA TCCAGACCGC CTTTAATCAA GGCGCTGGGA CTTGCACCCT 15163
GTCAGATTCA GGGAGGATAA GTTACACTGT GGAGTTTAGT TTGCCTACGC ATCATACTGT 15223
GCGCCTGATC CGCGTCACAG CATCACCCTC AGCATGATGG GCTGGCATTC TTGAGGCATC 15283
TCAGTGTTTG AATTGGAAGA ATGTGTGGTG AATGGCACTG ATTGACATTG TGCCTCTAAG 15343
TCACCTATTC AATTAGGGCG ACCGTGTGGG GGTGAGATTT AATTGGCGAG AACCATGCGG 15403
CCGAAATT 15411
2) INFORMATION FOR SEQ TD N0:29:
(i) SEQUENCE CHARACTERISTICS:
37
CA 02418780 2003-02-28
(A) LENGTH:256 amino acids
(B) TYPE: Amino Acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Protein
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Sus scrofa
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:29:
Met Lys Trp Gly Pro Cys Lys Ala Phe Leu Thr Lys Leu Ala Asn Phe
1 5 10 15
Leu Trp Met Leu Ser Arg Ser Ser Trp Cys Pro Leu Leu Ile Ser Leu
20 25 30
Tyr Phe Trp Pro Phe Cys Leu Ala Ser Pro Ser Pro Val Gly Trp Trp
35 40 45
Ser Phe Ala Ser Asp Trp Phe Ala Pro Arg Tyr Ser Val Arg Ala Leu
50 55 60
Pro Phe Thr Leu Ser Asn Tyr Arg Arg Ser Tyr Glu Ala Phe Leu Ser
65 70 75 80
Gln Cys Gln Val Asp Ile Pro Thr Trp Gly Thr Lys His Pro Leu Gly
85 90 95
Met Leu Trp His His Lys Val Ser Thr Leu Ile Asp Glu Met Val Ser
100 105 110
Arg Arg Met Tyr Arg Ile Met Glu Lys Ala Gly Gln Ala Ala Trp Lys
115 120 125
Gln Val Val Ser Glu Ala Thr Leu Ser Arg Ile Ser Ser Leu Asp Val
130 135 140
Val Ala His Phe Gln His Leu Ala Ala Ile Glu Ala Glu Thr Cys Lys
145 150 155 160
Tyr Leu Ala Ser Arg Leu Pro Met Leu His Asn Leu Arg Met Thr Gly
165 170 175
Ser Asn Val Thr Ile Val Tyr Asn Ser Thr Leu Asn Gln Val Phe Ala
180 185 190
Ile Phe Pro Thr Pro Gly Ser Arg Pro Lys Leu His Asp Phe Gln Gln
195 200 205
Trp Leu Ile Ala Val His Ser Ser Ile Phe Ser Ser Val Ala Ala Ser
210 215 220
Cys Thr Leu Phe Val Val Leu Trp Leu Arg Val Pro Ile Leu Arg Thr
225 230 235 240
Val Phe Gly Phe Arg Trp Leu Gly Ala Ile Phe Leu Ser Asn Ser Gln
245 250 255
(2) INFORMATION FOR SEQ ID N0:30:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15411 base pairs
(B) TYPE: Nucleic acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(ii) MOLECULE TYPE: DNA
(vi) ORIGINAL SOURCE:
(A) ORGANISM:Sus scrofa
(1x) FEATURE:
(A) NAME/KEY:CDS
(B) LOCATION: (13241)...(13777)
38
CA 02418780 2003-02-28
(D) OTHER INFORMATION: wt0RF4 of PRRSV strain VR-2332 GenBank
Accession #U87392
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:30:
ATGACGTATA GGTGTTGGCT CTATGCCTTG GCATTTGTAT TGTCAGGAGC TGTGACCATT 60
GGCACAGCCC AAAACTTGCT GCACAGAAAC ACCCTTCTGT GATAGCCTCC TTCAGGGGAG 120
CTTAGGGTTT GTCCCTAGCA CCTTGCTTCC GGAGTTGCAC TGCTTTACGG TCTCTCCACC 180
CCTTTAACCA TGTCTGGGAT ACTTGATCGG TGCACGTGTA CCCCCAATGC CAGGGTGTTT 240
ATGGCGGAGG GCCAAGTCTA CTGCACACGA TGCCTCAGTG CACGGTCTCT CCTTCCCCTG 300
AACCTCCAAG TTTCTGAGCT CGGGGTGCTA GGCCTATTCT ACAGGCCCGA AGAGCCACTC 360
CGGTGGACGT TGCCACGTGC ATTCCCCACT GTTGAGTGCT CCCCCGCCGG GGCCTGCTGG 420
CTTTCTGCAA TCTTTCCAAT CGCACGAATG ACCAGTGGAA ACCTGAACTT CCAACAAAGA 480
ATGGTACGGG TCGCAGCTGA GCTTTACAGA GCCGGCCAGC TCACCCCTGC AGTCTTGAAG 540
GCTCTACAAG TTTATGAACG GGGTTGCCGC TGGTACCCCA TTGTTGGACC TGTCCCTGGA 600
GTGGCCGTTT TCGCCAATTC CCTACATGTG AGTGATAAAC CTTTCCCGGG AGCAACTCAC 660
GTGTTGACCA ACCTGCCGCT CCCGCAGAGA CCCAAGCCTG AAGACTTTTG CCCCTTTGAG 720
TGTGCTATGG CTACTGTCTA TGACATTGGT CATGACGCCG TCATGTATGT GGCCGAAAGG 780
AAAGTCTCCT GGGCCCCTCG TGGCGGGGAT GAAGTGAAAT TTGAAGCTGT CCCCGGGGAG 840
TTGAAGTTGA TTGCGAACCG GCTCCGCACC TCCTTCCCGC CCCACCACAC AGTGGACATG 900
TCTAAGTTCG CCTTCACAGC CCCTGGGTGT GGTGTTTCTA TGCGGGTCGA ACGCCAACAC 960
GGCTGCCTTC CCGCTGACAC TGTCCCTGAA GGCAACTGCT GGTGGAGCTT GTTTGACTTG 1020
CTTCCACTGG AAGTTCAGAA CAAAGAAATT CGCCATGCTA ACCAATTTGG CTACCAGACC 1080
AAGCATGGTG TCTCTGGCAA GTACCTACAG CGGAGGCTGC AAGTTAATGG TCTCCGAGCA 1140
GTAACTGACC TAAACGGACC TATCGTCGTA CAGTACTTCT CCGTTAAGGA GAGTTGGATC 1200
CGCCATTTGA AACTGGCGGG AGAACCCAGC TACTCTGGGT TTGAGGACCT CCTCAGAATA 1260
AGGGTTGAGC CTAACACGTC GCCATTGGCT GACAAGGAAG AAAAAATTTT CCGGTTTGGC 1320
AGTCACAAGT GGTACGGCGC TGGAAAGAGA GCAAGAAAAG CACGCTCTTG TGCGACTGCT 1380
ACAGTCGCTG GCCGCGCTTT GTCCGTTCGT GAAACCCGGC AGGCCAAGGA GCACGAGGTT 1440
GCCGGCGCCA ACAAGGCTGA GCACCTCAAA CACTACTCCC CGCCTGCCGA AGGGAATTGT 1500
GGTTGGCACT GCATTTCCGC CATCGCCAAC CGGATGGTGA ATTCCAAATT TGAAACCACC 1560
CTTCCCGAAA GAGTGAGACC TCCAGATGAC TGGGCTACTG ACGAGGATCT TGTGAATGCC 1620
ATCCAAATCC TCAGACTCCC TGCGGCCTTA GACAGGAACG GTGCTTGTAC TAGCGCCAAG 1680
TACGTACTTA AGCTGGAAGG TGAGCATTGG ACTGTCACTG TGACCCCTGG GATGTCCCCT 1740
TCTTTGCTCC CTCTTGAATG TGTTCAGGGC TGTTGTGGGC ACAAGGGCGG TCTTGGTTCC 1800
CCAGATGCAG TCGAGGTCTC CGGATTTGAC CCTGCCTGCC TTGACCGGCT GGCTGAGGTG 1860
ATGCACCTGC CTAGCAGTGC TATCCCAGCC GCTCTGGCCG AAATGTCTGG CGATTCCGAT 1920
CGTTCGGCTT CTCCGGTCAC CACCGTGTGG ACTGTTTCGC AGTTCTTTGC CCGTCACAGC 1980
GGAGGGAATC ACCCTGACCA AGTGCGCTTA GGGAAAATTA TCAGCCTTTG TCAGGTGATT 2040
GAGGACTGCT GCTGTTCCCA GAACAAAACC AACCGGGTCA CCCCGGAGGA GGTCGCAGCA 2100
AAGATTGACC TGTACCTCCG TGGTGCAACA AATCTTGAAG AATGCTTGGC CAGGCTTGAG 2160
AAAGCGCGCC CGCCACGCGT AATCGACACC TCCTTTGATT GGGATGTTGT GCTCCCTGGG 2220
GTTGAGGCGG CAACCCAGAC GATCAAGCTG CCCCAGGTCA ACCAGTGTCG TGCTCTGGTC 2280
CCTGTTGTGA CTCAAAAGTC CTTGGACAAC AACTCGGTCC CCCTGACCGC CTTTTCACTG 2340
GCTAACTACT ACTACCGTGC GCAAGGTGAC GAAGTTCGTC ACCGTGAAAG ACTAACCGCC 2400
GTGCTCTCCA AGTTGGAAAA GGTTGTTCGA GAAGAATATG GGCTCATGCC AACCGAGCCT 2460
GGTCCACGGC CCACACTGCC ACGCGGGCTC GACGAACTCA AAGACCAGAT GGAGGAGGAC 2520
TTGCTGAAAC TGGCTAACGC CCAGACGACT TCGGACATGA TGGCCTGGGC AGTCGAGCAG 2580
GTTGACCTAA AAACTTGGGT CAAGAACTAC CCGCGGTGGA CACCACCACC CCCTCCGCCA 2640
AAAGTTCAGC CTCGAAAAAC GAAGCCTGTC AAGAGCTTGC CGGAGAGAAA GCCTGTCCCC 2700
GCCCCGCGCA GGAAGGTTGG GTCCGATTGT GGCAGCCCGG TTTCATTAGG CGGCGATGTC 2760
CCTAACAGTT GGGAAGATTT GGCTGTTAGT AGCCCCTTTG ATCTCCCGAC CCCACCTGAG 2820
CCGGCAACAC CTTCAAGTGA GCTGGTGATT GTGTCCTCAC CGCAATGCAT CTTCAGGCCG 2880
GCGACACCCT TGAGTGAGCC GGCTCCAATT CCCGCACCTC GCGGAACTGT GTCTCGACCG 2940
GTGACACCCT TGAGTGAGCC GATCCCTGTG CCCGCACCGC GGCGTAAGTT TCAGCAGGTG 3000
AAAAGATTGA GTTCGGCGGC GGCAATCCCA CCGTACCAGG ACGAGCCCCT GGATTTGTCT 3060
GCTTCCTCAC AGACTGAATA TGAGGCCTCT CCCCCAGCAC CGCCGCAGAG CGGGGGCGTT 3120
CTGGGAGTAG AGGGGCATGA AGCTGAGGAA ACCCTGAGTG AAATCTCGGA CATGTCGGGT 3180
AACATTAAAC CTGCGTCCGT GTCATCAAGC AGCTCCTTGT CCAGCGTGAG AATCACACGC 3240
CCAAAATACT CAGCTCAAGC CATCATCGAC TCGGGCGGGC CCTGCAGTGG GCATCTCCAA 3300
GAGGTAAAGG AAACATGCCT TAGTGTCATG CGCGAGGCAT GTGATGCGAC TAAGCTTGAT 3360
GACCCTGCTA CGCAGGAATG GCTTTCTCGC ATGTGGGATC GGGTGGACAT GCTGACTTGG 3420
39
CA 02418780 2003-02-28
CGCAACACGT CTGTTTACCA GGCGATTTGC ACCTTAGATG GCAGGTTAAA GTTCCTCCCA 3480
AAAATGATAC TCGAGACACC GCCGCCCTAT CCGTGTGAGT TTGTGATGAT GCCTCACACG 3540
CCTGCACCTT CCGTAGGTGC GGAGAGCGAC CTTACCATTG GCTCAGTTGC TACTGAAGAT 3600
GTTCCACGCA TCCTCGAGAA AATAGAAAAT GTCGGCGAGA TGGCCAACCA GGGACCCTTG 3660
GCCTTCTCCG AGGATAAACC GGTAGATGAC CAACTTGTCA ACGACCCCCG GATATCGTCG 3720
CGGAGGCCTG ACGAGAGCAC ATCAGCTCCG TCCGCAGGCA CAGGTGGCGC CGGCTCTTTT 3780
ACCGATTTGC CGCCTTCAGA TGGCGCGGAT GCGGACGGGG GGGGGCCGTT TCGGACGGTA 3840
AAAAGAAAAG CTGAAAGGCT CTTTGACCAA CTGAGCCGTC AGGTTTTTGA CCTCGTCTCC 3900
CATCTCCCTG TTTTCTTCTC ACGCCTTTTC TACCCTGGCG GTGGTTATTC TCCGGGTGAT 3960
TGGGGTTTTG CAGCTTTTAC TCTATTGTGC CTCTTTTTAT GTTACAGTTA CCCAGCCTTT 4020
GGTATTGCTC CCCTCTTGGG TGTGTTTTCT GGGTCTTCTC GGCGCGTTCG AATGGGGGTT 4080
TTTGGCTGCT GGTTGGCTTT TGCTGTTGGT CTGTTCAAGC CTGTGTCCGA CCCAGTCGGC 4140
GCTGCTTGTG AGTTTGACTC GCCAGAGTGT AGAAACATCC TTCATTCTTT TGAGCTTCTC 4200
AAACCTTGGG ACCCTGTTCG CAGCCTTGTT GTGGGCCCCG TCGGTCTCGG TCTTGCCATT 4260
CTTGGCAGGT TACTGGGCGG GGCACGCTGC ATCTGGCACT TTTTGCTTAG GCTTGGCATT 4320
GTTGCAGACT GTATCTTGGC TGGAGCTTAC GTGCTTTCTC AAGGTAGGTG TAAAAAGTGC 4380
TGGGGATCTT GTATAAGAAC TGCTCCTAAT GAGGTCGCTT TTAACGTGTT TCCTTTCACA 4440
CGTGCGACCA GGTCGTCACT TATCGACCTG TGCGATCGGT TTTGTGCGCC AAAAGGAATG 4500
GACCCCATTT TTCTCGCCAC TGGGTGGCGC GGGTGCTGGG CCGGCCGAAG CCCCATTGAG 4560
CAACCCTCTG AAAAACCCAT CGCGTTTGCC CAATTGGATG AAAAGAAGAT TACGGCTAGG 4620
ACTGTGGTCG CCCAGCCTTA TGACCCCAAC CAAGCCGTAA AGTGCTTGCG GGTATTGCAG 4680
TCGGGTGGGG CGATGGTGGC TAAGGCGGTC CCAAAAGTGG TCAAGGTTTC CGCTGTTCCA 4740
TTCCGAGCCC CCTTCTTTCC CACTGGAGTG AAAGTTGACC CTGATTGCAG GGTCGTGGTT 4800
GACCCTGACA CTTTCACTGC AGCTCTCCGG TCTGGCTACT CCACCACAAA CCTCGTCCTT 4860
GGTGTAGGGG ACTTTGCCCA GCTGAATGGA TTAAAAATCA GGCAAATTTC CAAGCCTTCA 4920
GGGGGAGGCC CACATCTCAT GGCTGCCCTG CATGTTGCCT GCTCGATGGC TCTGCACATG 4980
CTTGCTGGGA TTTATGTGAC TGCGGTGGGT TCTTGCGGCA CCGGCACCAA CGACCCGTGG 5040
TGCGCTAACC CGTTTGCCGT CCCTGGCTAC GGACCTGGCT CTCTCTGCAC GTCCAGGTTG 5100
TGCATTTCCC AACACGGCCT TACCCTGCCC TTGACAGCAC TTGTGGCGGG ATTCGGTATT 5160
CAAGAAATTG CCTTGGTCGT TTTGATTTTT GTTTCCATCG GAGGCATGGC TCATAGGTTG 5220
AGCTGTAAGG CTGACATGCT GTGTGTTTTG CTTGCAATTG CCAGCTATGT TTGGGTACCT 5280
CTTACCTGGT TGCTTTGTGT GTTTCCTTGC TGGTTGCGCT GTTTTTCTTT GCACCCCCTC 5340
ACCATCCTAT GGTTGGTGTT TTTCTTGATT TCTGTGAATA TGCCTTCAGG AATCTTGGCC 5400
ATGGTGTTGT TGGTTTCTCT TTGGCTTCTT GGTCGTTATA CTAATGTTGC TGGCCTTGTC 5460
ACCCCCTACG ACATTCATCA TTACACCAGT GGCCCCCGCG GTGTTGCCGC CTTGGCTACC 5520
GCACCAGATG GGACCTACTT GGCCGCTGTC CGCCGCGCTG CGTTGACTGG CCGCACCATG 5580
CTGTTTACCC CGTCCCAGCT TGGGTCTCTT CTTGAGGGTG CTTTCAGAAC TCGAAAGCCC 5640
TCACTGAACA CCGTCAATGT GATCGGGTCC TCCATGGGCT CTGGCGGGGT GTTTACCATC 5700
GACGGGAAAG TCAAGTGCGT AACTGCCGCA CATGTCCTTA CGGGCAATTC AGCTCGGGTT 5760
TCCGGGGTCG GCTTCAATCA AATGCTTGAC TTTGACGTAA AGGGAGATTT CGCTATAGCT 5820
GATTGCCCGA ATTGGCAAGG GGCTGCCCCC AAGACCCAAT TCTGCACGGA TGGATGGACT 5880
GGCCGTGCCT ATTGGCTAAC ATCCTCTGGC GTCGAACCCG GCGTCATTGG AAAAGGATTC 5940
GCCTTCTGCT TCACCGCATG TGGCGATTCC GGGTCCCCAG TGATCACCGA GGCCGGTGAG 6000
CTTGTCGGCG TTCACACGGG ATCGAATAAA CAAGGGGGGG GCATTGTTAC GCGCCCCTCA 6060
GGCCAGTTTT GTAATGTGGC ACCCATCAAG CTAAGCGAAT TAAGTGAATT CTTTGCTGGG 6120
CCTAAGGTCC CGCTCGGTGA TGTGAAGGTC GGCAGCCACA TAATTAAAGA CATAAGCGAG 6180
GTGCCTTCAG ATCTTTGTGC CTTGCTTGCT GCCAAACCTG AACTGGAAGG AGGCCTCTCC 6240
ACCGTCCAAC TTCTTTGTGT GTTTTTTCTC CTGTGGAGAA TGATGGGACA TGCCTGGACG 6300
CCCTTGGTTG CTGTGAGTTT CTTTATTTTG AATGAGGTTC TCCCAGCCGT CCTGGTCCGG 6360
AGTGTTTTCT CCTTTGGAAT GTTTGTGCTA TCCTGGCTCA CGCCATGGTC TGCGCAAGTT 6420
CTGATGATCA GGCTTCTGAC AGCAGCTCTT AACAGGAACA GATGGTCACT TGCCTTTTTC 6480
AGCCTCGGTG CAGTGACCGG TTTTGTCGCA GATCTTGCGG CCACTCAGGG GCATCCGTTG 6540
CAGGCAGTGA TGAATTTGAG CACCTATGCA TTCCTGCCTC GGATGATGGT TGTGACCTCA 6600
CCAGTCCCAG TGATCACGTG TGGTGTCGTG CACCTACTTG CCATCATTTT GTACTTGTTT 6660
AAGTACCGTG GCCCGCACCA TATCCTTGTT GGCGATGGAG TGTTCTCTGC GGCTTTCTTC 6720
TTGAGATACT TTGCCGAGGG AAAGTTGAGG GAAGGGGTGT CGCAATCCTG CGGAATGAAT 6780
CATGAGTCTC TGACTGGTGC CCTCGCTATG AGACTCAATG ACGAGGACTT GGATTTCCTT 6840
ATGAAATGGA CTGATTTTAA GTGCTTTGTT TCTGCGTCCA ACATGAGGAA TGCAGCGGGT 6900
CAATTTATCG AGGCTGCCTA TGCTAAAGCA CTTAGAGTAG AACTGGCCCA GTTGGTGCAG 6960
GTTGATAAAG TTCGAGGTAC TTTGGCCAAA CTTGAAGCTT TTGCTGATAC CGTGGCACCT 7020
CAACTCTCGC CCGGTGACAT TGTTGTCGCT CTCGGCCACA CGCCTGTTGG CAGTATCTTC 7080
GACCTAAAGG TTGGTAGCAC CAAGCATACC CTCCAAGCCA TTGAGACCAG AGTCCTTGCT 7140
CA 02418780 2003-02-28
GGGTCCAAAA TGACCGTGGC GCGCGTCGTC GACCCGACCC CCACGCCCCC ACCCGCACCC 7200
GTGCCCATCC CCCTCCCACC GAAAGTTCTG GAGAATGGCC CCAACGCTTG GGGGGATGAG 7260
GACCGTTTGA ATAAGAAGAA GAGGCGCAGG ATGGAAGCCC TCGGCATCTA TGTTATGGGC 7320
GGGAAAAAGT ACCAGAAATT TTGGGACAAG AATTCCGGTG ATGTGTTTTA TGAGGAGGTC 7380
CATAATAACA CAGATGAGTG GGAGTGTCTC AGAGTTGGCG ACCCTGCCGA CTTTGACCCT 7440
GAGAAGGGAA CTCTGTGTGG ACATGTCACC ATTGAAAACA AGGCTTACCA TGTTTACACC 7500
TCCCCATCTG GTAAGAAGTT CTTGGTCCCC GTCAACCCAG AGAATGGAAG AGTTCAATGG 7560
GAAGCTGCAA AGCTTTCCGT GGAGCAGGCC CTAGGTATGA TGAATGTCGA CGGCGAACTG 7620
ACTGCCAAAG AACTGGAGAA ACTGAAAAGA ATAATTGACA AACTCCAGGG CCTGACTAAG 7680
GAGCAGTGTT TAAACTGCTA GCCGCCAGCG ACTTGACCCG CTGTGGTCGC GGCGGCTTGG 7740
TTGTTACTGA AACAGCGGTA AAAATAGTCA AATTTCACAA CCGGACCTTC ACCCTGGGAC 7800
CTGTGAATTT AAAAGTGGCC AGTGAGGTTG AGCTAAAAGA CGCGGTTGAG CACAACCAAC 7860
ACCCGGTTGC GAGACCGATC GATGGTGGAG TTGTGCTCCT GCGTTCCGCG GTTCCTTCGC 7920
TTATAGACGT CTTGATCTCC GGTGCTGATG CATCTCCCAA GTTACTTGCC CATCACGGGC 7980
CGGGAAACAC TGGGATCGAT GGCACGCTCT GGGATTTTGA GTCCGAAGCC ACTAAAGAGG 8040
AAGTCGCACT CAGTGCGCAA ATAATACAGG CTTGTGACAT TAGGCGCGGC GACGCTCCTG 8100
AAATTGGTCT CCCTTACAAG CTGTACCCTG TTAGGGGTAA CCCTGAGCGG GTGAAAGGAG 8160
TTCTGCAGAA TACAAGGTTT GGAGACATAC CTTACAAAAC CCCCAGTGAC ACTGGAAGCC 8220
CAGTGCACGC GGCTGCCTGC CTTACGCCCA ACGCCACTCC GGTGACTGAT GGGCGCTCCG 8280
TCTTGGCCAC GACCATGCCC CCCGGGTTTG AGTTATATGT ACCGACCATA CCAGCGTCTG 8340
TCCTTGATTA CCTTGACTCT AGGCCTGACT GCCCTAAACA GCTGACAGAG CACGGCTGCG 8400
AAGATGCCGC ACTGAAAGAC CTCTCTAAAT ATGACTTGTC CACCCAAGGC TTTGTTTTAC 8460
CTGGAGTTCT TCGCCTTGTG CGGAAATACC TGTTTGCCCA TGTAGGTAAG TGCCCACCCG 8520
TTCATCGGCC TTCTACTTAC CCTGCTAAGA ATTCTATGGC TGGAATAAAT GGGAACAGGT 8580
TCCCAACCAA GGACATTCAG AGCGTCCCTG AAATCGACGT TCTGTGCGCA CAGGCTGTGC 8640
GAGAAAACTG GCAAACTGTC ACCCCTTGTA CTCTTAAGAA ACAGTATTGC GGGAAGAAGA 8700
AGACTAGGAC CATACTCGGC ACCAATAACT TCATCGCACT AGCCCACCGA GCAGTGTTGA 8760
GTGGTGTTAC CCAGGGCTTC ATGAAAAAGG CGTTTAACTC GCCCATCGCC CTCGGAAAGA 8820
ACAAGTTTAA GGAGCTACAG ACTCCGGTCC TGGGCAGGTG CCTTGAAGCT GATCTCGCAT 8880
CCTGCGATCG ATCCACGCCT GCAATTGTCC GCTGGTTTGC CGCCAACCTT CTTTATGAAC 8940
TTGCCTGTGC TGAAGAGCAT CTACCGTCGT ACGTGCTGAA CTGCTGCCAC GACTTACTGG 9000
TCACGCAGTC CGGCGCAGTG ACTAAGAGAG GTGGCCTGTC GTCTGGCGAC CCGATCACCT 9060
CTGTGTCTAA CACCATTTAT AGTTTGGTGA TCTATGCACA GCATATGGTG CTTAGTTACT 9120
TCAAAAGTGG TCACCCCCAT GGCCTTCTGT TCTTACAAGA CCAGCTAAAG TTTGAGGACA 9180
TGCTCAAGGT TCAACCCCTG ATCGTCTATT CGGACGACCT CGTGCTGTAT GCCGAGTCTC 9240
CCACCATGCC AAACTATCAC TGGTGGGTTG AACATCTGAA TTTGATGCTG GGGTTTCAGA 9300
CGGACCCAAA GAAGACAGCA ATAACAGACT CGCCATCATT TCTAGGCTGT AGAATAATAA 9360
ATGGGCGCCA GCTAGTCCCC AACCGTGACA GGATCCTCGC GGCCCTCGCC TATCACATGA 9420
AGGCGAGTAA TGTTTCTGAA TACTATGCCT CAGCGGCTGC AATACTCATG GACAGCTGTG 9480
CTTGTTTGGA GTATGATCCT GAATGGTTTG AAGAACTTGT AGTTGGAATA GCGCAGTGCG 9540
CCCGCAAGGA CGGCTACAGC TTTCCCGGCA CGCCGTTCTT CATGTCCATG TGGGAAAAAC 9600
TCAGGTCCAA TTATGAGGGG AAGAAGTCGA GAGTGTGCGG GTACTGCGGG GCCCCGGCCC 9660
CGTACGCTAC TGCCTGTGGC CTCGACGTCT GCATTTACCA CACCCACTTC CACCAGCATT 9720
GTCCAGTCAC AATCTGGTGT GGCCATCCAG CGGGTTCTGG TTCTTGTAGT GAGTGCAAAT 9780
CCCCTGTAGG GAAAGGCACA AGCCCTTTAG ACGAGGTGCT GGAACAAGTC CCGTATAAGC 9840
CCCCACGGAC CGTTATCATG CATGTGGAGC AGGGTCTCAC CCCCCTTGAT CCAGGTAGAT 9900
ACCAAACTCG CCGCGGATTA GTCTCTGTCA GGCGTGGAAT TAGGGGAAAT GAAGTTGGAC 9960
TACCAGACGG TGATTATGCT AGCACCGCCT TGCTCCCTAC CTGCAAAGAG ATCAACATGG 10020
TCGCTGTCGC TTCCAATGTA TTGCGCAGCA GGTTCATCAT CGGCCCACCC GGTGCTGGGA 10080
AAACATACTG GCTCCTTCAA CAGGTCCAGG ATGGTGATGT TATTTACACA CCAACTCACC 10140
AGACCATGCT TGACATGATT AGGGCTTTGG GGACGTGCCG GTTCAACGTC CCGGCAGGCA 10200
CAACGCTGCA ATTCCCCGTC CCCTCCCGCA CCGGTCCGTG GGTTCGCATC CTAGCCGGCG 10260
GTTGGTGTCC TGGCAAGAAT TCCTTCCTAG ATGAAGCAGC GTATTGCAAT CACCTTGATG 10320
TTTTGAGGCT TCTTAGTAAA ACTACCCTCA CCTGTCTAGG AGACTTCAAG CAACTCCACC 10380
CAGTGGGTTT TGATTCTCAT TGCTATGTTT TTGACATCAT GCCTCAAACT CAACTGAAGA 10440
CCATCTGGAG GTTTGGACAG AATATCTGTG ATGCCATTCA GCCAGATTAC AGGGACAAAC 10500
TCATGTCCAT GGTCAACACA ACCCGTGTGA CCTACGTGGA AAAACCTGTC AGGTATGGGC 10560
AGGTCCTCAC CCCCTACCAC AGGGACCGAG AGGACGACGC CATCACTATT GACTCCAGTC 10620
AAGGCGCCAC ATTCGATGTG GTTACATTGC ATTTGCCCAC TAAAGATTCA CTCAACAGGC 10680
AAAGAGCCCT TGTTGCTATC ACCAGGGCAA GACACGCTAT CTTTGTGTAT GACCCACACA 10740
GGCAGCTGCA GGGCTTGTTT GATCTTCCTG CAAAAGGCAC GCCCGTCAAC CTCGCAGTGC 10800
ACTGCGACGG GCAGCTGATC GTGCTGGATA GAAATAACAA AGAATGCACG GTTGCTCAGG 10860
4t
CA 02418780 2003-02-28
CTCTAGGCAA CGGGGATAAA TTTAGGGCCA CAGACAAGCG TGTTGTAGAT TCTCTCCGCG 10920
CCATTTGTGC TGATCTAGAA GGGTCGAGCT CTCCGCTCCC CAAGGTCGCA CACAACTTGG 10980
GATTTTATTT CTCACCTGAT TTAACACAGT TTGCTAAACT CCCAGTAGAA CTTGCACCTC 11040
ACTGGCCCGT GGTGTCAACC CAGAACAATG AAAAGTGGCC GGATCGGCTG GTTGCCAGCC 11100
TTCGCCCTAT CCATAAATAC AGCCGCGCGT GCATCGGTGC CGGCTATATG GTGGGCCCTT 11160
CGGTGTTTCT AGGCACTCCT GGGGTCGTGT CATACTATCT CACAAAATTT GTTAAGGGCG 11220
GGGCTCAAGT GCTTCCGGAG ACGGTTTTCA GCACCGGCCG AATTGAGGTA GACTGCCGGG 11280
AATATCTTGA TGATCGGGAG CGAGAAGTTG CTGCGTCCCT CCCACACGGT TTCATTGGCG 11340
ACGTCAAAGG CACTACCGTT GGAGGATGTC ATCATGTCAC CTCCAGATAC CTCCCGCGCG 11400
TCCTTCCCAA GGAATCAGTT GCGGTAGTCG GGGTTTCAAG CCCCGGAAAA GCCGCGAAAG 11460
CATTGTGCAC ACTGACAGAT GTGTACCTCC CAGATCTTGA AGCCTATCTC CACCCGGAGA 11520
CCCAGTCCAA GTGCTGGAAA ATGATGTTGG ACTTCAAAGA AGTTCGACTA ATGGTCTGGA 11580
AAGACAAAAC AGCCTATTTC CAACTTGAAG GTCGCTATTT CACCTGGTAT CAGCTTGCCA 11640
GCTATGCCTC GTACATCCGT GTTCCCGTCA ACTCTACGGT GTACTTGGAC CCCTGCATGG 11700
GCCCCGCCCT TTGCAACAGG AGAGTCGTCG GGTCCACCCA CTGGGGGGCT GACCTCGCGG 11760
TCACCCCTTA TGATTACGGC GCTAAAATTA TCCTGTCTAG CGCGTACCAT GGTGAAATGC 11820
CCCCCGGATA CAAAATTCTG GCGTGCGCGG AGTTCTCGTT GGATGACCCA GTTAAGTACA 11880
AACATACCTG GGGGTTTGAA TCGGATACAG CGTATCTGTA TGAGTTCACC GGAAACGGTG 11940
AGGACTGGGA GGATTACAAT GATGCGTTTC GTGCGCGCCA GGAAGGGAAA ATTTATAAGG 12000
CCACTGCCAC CAGCTTGAAG TTTTATTTTC CCCCGGGCCC TGTCATTGAA CCAACTTTAG 12060
GCCTGAATTG AAATGAAATG GGGTCCATGC AAAGCCTTTT TGACAAAATT GGCCAACTTT 12120
TTGTGGATGC TTTCACGGAG TTCTTGGTGT CCATTGTTGA TATCATTATA TTTTTGGCCA 12180
TTTTGTTTGG CTTCACCATC GCCGGTTGGC TGGTGGTCTT TTGCATCAGA TTGGTTTGCT 12240
CCGCGATACT CCGTACGCGC CCTGCCATTC ACTCTGAGCA ATTACAGAAG ATCTTATGAG 12300
GCCTTTCTTT CCCAGTGCCA AGTGGACATT CCCACCTGGG GAACTAAACA TCCTTTGGGG 12360
ATGCTTTGGC ACCATAAGGT GTCAACCCTG ATTGATGAAA TGGTGTCGCG TCGAATGTAC 12420
CGCATCATGG AAAAAGCAGG GCAGGCTGCC TGGAAACAGG TGGTGAGCGA GGCTACGCTG 12480
TCTCGCATTA GTAGTTTGGA TGTGGTGGCT CATTTTCAGC ATCTAGCCGC CATTGAAGCC 12540
GAGACCTGTA AATATTTGGC CTCCCGGCTG CCCATGCTAC ACAACCTGCG CATGACAGGG 12600
TCAAATGTAA CCATAGTGTA TAATAGCACT TTGAATCAGG TGTTTGCTAT TTTTCCAACC 12660
CCTGGTTCCC GGCCAAAGCT TCATGATTTT CAGCAATGGT TAATAGCTGT ACATTCCTCC 12720
ATATTTTCCT CTGTTGCAGC TTCTTGTACT CTTTTTGTTG TGCTGTGGTT GCGGGTTCCA 12780
ATACTACGTA CTGTTTTTGG TTTCCGCTGG TTAGGGGCAA TTTTTCTTTC GAACTCACAG 12840
TGAATTACAC GGTGTGTCCA CCTTGCCTCA CCCGGCAAGC AGCCACAGAG ATCTACGAAC 12900
CCGGTAGGTC TCTTTGGTGC AGGATAGGGT ATGACCGATG TGGGGAGGAC GATCATGACG 12960
AGCTAGGGTT TATGATACCG CCTGGCCTCT CCAGCGAAGG CCACTTGACT GGTGTTTACG 13020
CCTGGTTGGC GTTCTTGTCC TTCAGCTACA CGGCCCAGTT CCATCCCGAG ATATTCGGGA 13080
TAGGGAATGT GAGTCGAGTT TATGTTGACA TCAAACATCA ACTCATCTGC GCCGAACATG 13140
ACGGGCAGAA CACCACCTTG CCTCGTCATG ACAACATTTC AGCCGTGTTT CAGACCTATT 13200
ACCAACATCA AGTCGACGGC GGCAATTGGT TTCACCTAGA ATG GCT TCG TCC CTT 13255
Met Ala Ser Ser Leu
1 5
CTTTTCCTC GTGGTTGGTTTT AAATGTCTC TTGGTTTCTCAG GCGTTC 13303
LeuPheLeu ValValGlyPhe LysCysLeu LeuValSerGln AlaPhe
10 15 20
GCCTGCAAA CCATGTTTCAGT TCGAGTCTT GCAGATATTAAG ACCAAC 13351
AlaCysLys ProCysPheSer SerSerLeu AlaAspIleLys ThrAsn
25 30 35
ACCACCGCA GCGGCAAGCTTT GCTGTCCTC CAAGACATCAGT TGCCTT 13399
ThrThrAla AlaAlaSerPhe AlaValLeu GlnAspIleSer CysLeu
40 45 50
AGGCATCGC GACTCGGCCTCT GAGGCGATT CGCAAAATCCCT CAGTGC 13447
ArgHisArg AspSerAIaSer GluAlaIle ArgLysIlePro GlnCys
55 60 65
CGTACGGCG ATAGGGACACCC GTGTATGTT ACCATCACAGCC AATGTG 13495
ArgThrAla IleGlyThrPro ValTyrVal ThrIleThrAla AsnVal
70 75 80 85
42
CA 02418780 2003-02-28
ACAGATGAG TATTTA CATTCTTCT GATCTCCTC ATGCTTTCT TCT 13543
AAT
ThrAspGlu AsnTyrLeu HisSerSer AspLeuLeu MetLeuSer 5er
90 95 100
TGCCTTTTC TATGCTTCT GAGATGAGT GAAAAGGGA TTTAAGGTG GTA 13591
CysLeuPhe TyrAlaSer GluMetSer GluLysGly PheLysVal Val
105 110 115
TTTGGCAAT GTGTCAGGC ATCGTGGCT GTGTGTGTC AATTTTACC AGC 13639
PheGlyAsn ValSerGly IleValAla ValCysVal AsnPheThr Ser
120 125 130
TACGTCCAA CATGTCAAG GAGTTTACC CAACGCTCC CTGGTGGTC GAC 13687
TyrValGln HisValLys GluPheThr GlnArgSer LeuValVal Asp
135 140 145
CATGTGCGG TTGCTCCAT TTCATGACA CCTGAGACC ATGAGGTGG GCA 13735
HisValArg LeuLeuHis PheMetThr ProGluThr MetArgTrp Ala
150 155 160 165
ACTGTTTTA GCCTGTCTT TTTGCCATT CTGTTGGCA ATTTGA 13777
ThrValLeu AlaCysLeu PheAlaIle LeuLeuAla Ile
170 175
ATGTTTAAGT ATGTTGGAGA AATGCTTGAC CGCGGGCTGT TGCTCGCGAT TGCTTTCTTT 13837
GTGGTGTATC GTGCCGTTCT GTTTTGCTGT GCTCGCCAAC GCCAGCAACG ACAGCAGCTC 13897
CCATCTACAG CTGATTTACA ACTTGACGCT ATGTGAGCTG AATGGCACAG ATTGGCTAGC 13957
TAACAAATTT GATTGGGCAG TGGAGAGTTT TGTCATCTTT CCCGTTTTGA CTCACATTGT 14017
CTCCTATGGT GCCCTCACTA CCAGCCATTT CCTTGACACA GTCGCTTTAG TCACTGTGTC 14077
TACCGCCGGG TTTGTTCACG GGCGGTATGT CCTAAGTAGC ATCTACGCGG TCTGTGCCCT 14137
GGCTGCGTTG ACTTGCTTCG TCATTAGGTT TGCAAAGAAT TGCATGTCCT GGCGCTACGC 14197
GTGTACCAGA TATACCAACT TTCTTCTGGA CACTAAGGGC AGACTCTATC GTTGGCGGTC 14257
GCCTGTCATC ATAGAGAAAA GGGGCAAAGT TGAGGTCGAA GGTCATCTGA TCGACCTCAA 14317
AAGAGTTGTG CTTGATGGTT CCGTGGCAAC CCCTATAACC AGAGTTTCAG CGGAACAATG 14377
GGGTCGTCCT TAGATGACTT CTGTCATGAT AGCACGGCTC CACAAAAGGT GCTTTTGGCG 14437
TTTTCTATTA CCTACACGCC AGTGATGATA TATGCCCTAA AGGTGAGTCG CGGCCGACTG 14497
CTAGGGCTTC TGCACCTTTT GATCTTCCTG AATTGTGCTT TCACCTTCGG GTACATGACT 14557
TTCGCGCACT TTCAGAGTAC AAATAAGGTC GCGCTCACTA TGGGAGCAGT AGTTGCACTC 14617
CTTTGGGGGG TGTACTCAGC CATAGAAACC TGGAAATTCA TCACCTCCAG ATGCCGTTTG 14677
TGCTTGCTAG GCCGCAAGTA CATTCTGGCC CCTGCCCACC ACGTTGAAAG TGCCGCACGG 14737
TTTCATCCGA TTGCGGCAAA TGATAACCAC GCATTTGTCG TCCGGCGTCC CGGCTCCACT 14797
ACGGTCAACG GCACATTGGT GCCCGGGTTA AAAAGCCTCG TGTTGGGTGG CAGAAAAGCT 14857
GTTAAACAGG GAGTGGTAAA CCTTGTCAAA TATGCCAAAT AACAACGGCA AGCAGCAGAA 14917
GAGAAAGAAG GGGGATGGCC AGCCAGTCAA TCAGCTGTGC CAGATGCTGG GTAAGATCAT 14977
CGCTCAGCAA AACCAGTCCA GAGGCAAGGG ACCGGGAAAG AAAAATAAGA AGAAAAACCC 15037
GGAGAAGCCC CATTTTCCTC TAGCGACTGA AGATGATGTC AGACATCACT TTACCCCTAG 15097
TGAGCGGCAA TTGTGTCTGT CGTCAATCCA GACCGCCTTT AATCAAGGCG CTGGGACTTG 15157
CACCCTGTCA GATTCAGGGA GGATAAGTTA CACTGTGGAG TTTAGTTTGC CTACGCATCA 15217
TACTGTGCGC CTGATCCGCG TCACAGCATC ACCCTCAGCA TGATGGGCTG GCATTCTTGA 15277
GGCATCTCAG TGTTTGAATT GGAAGAATGT GTGGTGAATG GCACTGATTG ACATTGTGCC 15337
TCTAAGTCAC CTATTCAATT AGGGCGACCG TGTGGGGGTG AGATTTAATT GGCGAGAACC 15397
ATGCGGCCGA AATT 15411
2) INFORMATION FOR SEQ ID N0:31:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH:178 amino acids
(B) TYPE: Amino Acid
(D) TOPOLOGY: linear
43
CA 02418780 2003-02-28
(ii) MOLECULE TYPE: Protein
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Sus scrofa
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:31:
Met Ala Ser Ser Leu Leu Phe Leu Val Val Gly Phe Lys Cys Leu Leu
1 5 10 15
Val Ser GIn Ala Phe Ala Cys Lys Pro Cys Phe Ser Ser Ser Leu Ala
20 25 30
Asp Ile Lys Thr Asn Thr Thr Ala Ala Ala Ser Phe Ala Val Leu Gln
35 40 45
Asp Ile Ser Cys Leu Arg His Arg Asp Ser Ala Ser Glu Ala Ile Arg
50 55 60
Lys Ile Pro Gln Cys Arg Thr Ala Ile Gly Thr Pro Val Tyr Val Thr
65 70 75 80
Ile Thr Ala Asn Val Thr Asp Glu Asn Tyr Leu His Ser Ser Asp Leu
85 90 95
Leu Met Leu Ser Ser Cys Leu Phe Tyr Ala Ser Glu Met Ser Glu Lys
100 105 110
Gly Phe Lys Val Val Phe Gly Asn Val Ser Gly Ile Val Ala Val Cys
115 120 125
Val Asn Phe Thr Ser Tyr Val Gln His Val Lys Glu Phe Thr Gln Arg
130 135 140
Ser Leu Val Val Asp His Val Arg Leu Leu His Phe Met Thr Pro Glu
145 150 155 160
Thr Met Arg Trp Ala Thr Val Leu Ala Cys Leu Phe Ala Ile Leu Leu
165 170 175
Ala Ile
(2) INFORMATION FOR SEQ ID N0:32:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 603 base pairs
(B) TYPE: Nucleic acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(ii) MOLECULE TYPE: DNA
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Sus scrofa
(ix) FEATURE:
(A) NAME/KEY:CDS
(B) LOCATION: (1)...(603)
(D) OTHER INFORMATION: wtORFS of PRRSV strain 98-37120-2 GenBank
Accession #AF339499
(ix) FEATURE:
(A) NAME/KEY:Misc feature
(B1 LOCATION: (0) . . . (0)
(D) OTHER INFORMATION: n = a, t, g,or c
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:32:
ATG TTG GAG AAA TGC TTG ACC GCG GGC TGT TGC TCG CAA TTG CCT TTT 48
Met Leu Glu Lys Cys Leu Thr Ala Gly Cys Cys Ser Gln Leu Pro Phe
1 5 10 15
TTG TGG TGT ATC GTG CCG TTC TGT TTT GCT GTG CTC GTC GAC GCC AGC 96
44
CA 02418780 2003-02-28
LeuTrpCys IleVal ProPheCysPhe AlaValLeu ValAspAla Ser
20 25 30
AACAACAAC AGCTCC CATCTACAGCTG ATTTACAAC TTGACGCTA TGT 144
AsnAsnAsn SerSer HisLeuGlnLeu IleTyrAsn LeuThrLeu Cys
35 40 45
GAGCTGAAT GGCACN GATTGGCTAGCT GAAAAATTT GATTGGGCA GTG 192
GluLeuAsn GlyThr AspTrpLeuAla GluLysPhe AspTrpAla Val
50 55 60
GAGAGTTTT GTCATC TTTCCTGTTTTG ACTCACATT GTTTCTTAT GGT 240
GluSerPhe ValIle PheProValLeu ThrHisIle ValSerTyr Gly
65 70 75 80
GCCCTCACC ACCAGT CATTTCCTTGAC ACAGTCGCT TTAGTCACT GTG 288
AlaLeuThr ThrSer HisPheLeuAsp ThrValAla LeuValThr Val
85 90 95
TCTACCGCC GGGTTT GTTCACGGGCGG TATGTCCTA AGTAGCATC TAC 336
SerThrAla GlyPhe ValHisGlyArg TyrValLeu SerSerIle Tyr
100 105 110
GCGGTCTGT GCCCTG GCTGCGTTGACT TGCTTCGTC ATTAGGTTT GCA 384
AlaValCys AlaLeu AlaAlaLeuThr CysPheVal IleArgPhe Ala
115 120 125
AAGAATTGC ATGTCC TGGCGCTACGCG TGTACCAGA TATACCAAC TTT 432
LysAsnCys MetSer TrpArgTyrAla CysThrArg TyrThrAsn Phe
130 135 140
CTTCTGGAC ACTAAG GGCAGACTCTAT CGTTGGCGG TCGCCTGTC ATC 480
LeuLeuAsp ThrLys GlyArgLeuTyr ArgTrpArg SerProVal Ile
145 150 155 160
ATAGAGAAA AGGGGC AAAGTTGAGGTC GAAGGTCAT CTGATTGAC CTC 528
IleGluLys ArgGly LysValGluVal GluGlyHis LeuIleAsp Leu
165 170 175
AAAAGAGTT GTGCTT GATGGTTCCGTG GCAACCCCT ATAACCAGA GTT 576
LysArgVal ValLeu AspGlySerVal AlaThrPro IleThrArg Val
180 185 190
TCAGCGGAA CAATGG GGTCGTCCTTAG 603
SerAlaGlu GlnTrp GlyArgPro
195 200
2) INFORMATION FOR SEQ ID N0:33:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH:200 amino acids
(B) TYPE: Amino Acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Protein
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Sus scrofa
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:33:
CA 02418780 2003-02-28
Met Leu Glu Lys Cys Leu Thr Ala Gly Cys Cys Sex Gln Leu Pro Phe
1 5 10 15
Leu Trp Cys Ile Val Pro Phe Cys Phe Ala Val Leu Val Asp Ala Ser
20 25 30
Asn Asn Asn Ser Ser His Leu Gln Leu IIe Tyr Asn Leu Thr Leu Cys
35 40 45
Glu Leu Asn Gly Thr Asp Trp Leu Ala Glu Lys Phe Asp Trp Ala Val
50 55 60
Glu Ser Phe Val Ile Phe Pro Val Leu Thr His Ile Val Ser Tyr Gly
65 70 75 80
Ala Leu Thr Thr Ser His Phe Leu Asp Thr Val Ala Leu Val Thr Val
85 90 95
Ser Thr Ala Gly Phe Val His Gly Arg Tyr Val Leu Ser Ser Ile Tyr
100 105 110
Ala Val Cys Ala Leu Ala Ala Leu Thr Cys Phe Val Ile Arg Phe Ala
115 120 125
Lys Asn Cys Met Ser Trp Arg Tyr Ala Cys Thr Arg Tyr Thr Asn Phe
130 135 140
Leu Leu Asp Thr Lys Gly Arg Leu Tyr Arg Trp Arg Ser Pro Val Ile
145 150 155 160
Ile Glu Lys Arg Gly Lys Val Glu Val Glu Gly His Leu Ile Asp Leu
165 170 175
Lys Arg Val Val Leu Asp Gly Ser Val Ala Thr Pro Ile Thr Arg Val
180 185 190
Ser Ala Glu Gln Trp Gly Arg Pro
195 200
(2) INFORMATION FOR SEQ ID N0:34:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2050 base pairs
(B) TYPE: Nucleic acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(ii) MOLECULE TYPE: DNA
(vi) ORIGINAL SOURCE:
(A) ORGANISM:Sus scrofa
(ix) FEATURE:
(A) NAME/KEY:CDS
(B) LOCATION: (426)...(1028)
(D) OTHER INFORMATION: wtORF5 of PRRSV strain VR-2385 GenBank
Accession #U03040
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:34:
GGCAGGCTTT GCTGTCCTCC AAGACATCAG TTGCCTTAGG CATCGCAACT CGGCCTCTGA 60
GGCGATTCGC AAAGTCCCTC AGTGCCGCAC GGCGATAGGG ACACCCGTGT ATATCACTGT 120
CACAGCCAAT GTTACCGATG AGAATTATTT GCATTCCTCT GATCTTCTCA TGCTTTCTTC 180
TTGCCTTTTC TATGCTTCTG AGATGAGTGA AAAGGGATTT AAGGTGGTAT TTGGCAATGT 240
GTCAGGCATC GTGGCAGTGT GCGTCAACTT CACCAGTTAC GTCCAACATG TCAAGGAATT 300
TACCCAACGT TCCTTGGTAG TTGACCATGT GCGGCTGCTC CATTTCATGA CGCCCGAGAC 360
CATGAGGTGG GCAACTGTTT TAGCCTGTCT TTTTGGCATT CTGTTGGCAA TTTGAATGTT 420
TAAGT ATG TTG GGG AAA TGC TTG ACC GCG GGC TGT TGC TCG CAA TTG CTT 470
Met Leu Gly Lys Cys Leu Thr Ala Gly Cys Cys Ser Gln Leu Leu
1 5 10 15
TTT TTG TGG TGT ATC GTG CCG TCT TGT TTT GTT GCG CTC GTC AGC GCC 518
Phe Leu Trp Cys Ile Val Pro Ser Cys Phe Val Ala Leu Val Ser Ala
46
CA 02418780 2003-02-28
20 25 30
AAC GGG AAC AGC GGC TCA AAT TTA CAG CTG ATT TAC AAC TTG ACG CTA 566
Asn Gly Asn Ser Gly Ser Asn Leu Gln Leu Ile Tyr Asn Leu Thr Leu
35 40 45
TGT GAG CTG AAT GGC ACA GAT TGG CTA GCT AAT AAA TTT GAC TGG GCA 614
Cys Glu Leu Asn Gly Thr Asp Trp Leu Ala Asn Lys Phe Asp Trp Ala
50 55 60
GTG GAG TGT TTT GTC ATT TTT CCT GTG TTG ACT CAC ATT GTC TCT TAT 662
Val Glu Cys Phe Val Tle Phe Pro Val Leu Thr His Ile Val Ser Tyr
65 70 75
GGT GCC CTC ACT ACT AGC CAT TTC CTT GAC ACA GTC GGT CTG GTC ACT 710
Gly Ala Leu Thr Thr Ser His Phe Leu Asp Thr Val Gly Leu Val Thr
80 85 90 95
GTG TCT ACC GCT GGG TTT GTT CAC GGG CGG TAT GTT CTG AGT AGC ATG 758
Val Ser Thr Ala Gly Phe Val His Gly Arg Tyr Val Leu Ser Ser Met
100 105 110
TAC GCG GTC TGT GCC CTG GCT GCG TTG ATT TGC TTC GTC ATT AGG CTT 806
Tyr Ala Val Cys Ala Leu Ala Ala Leu Ile Cys Phe Val Ile Arg Leu
115 120 125
GCG AAG AAT TGC ATG TCC TGG CGC TAC TCA TGT ACC AGA TAT ACC AAC 854
Ala Lys Asn Cys Met Ser Trp Arg Tyr Ser Cys Thr Arg Tyr Thr Asn
130 135 140
TTT CTT CTG GAC ACT AAG GGC AGA CTC TAT CGT TGG CGG TCG CCT GTC 902
Phe Leu Leu Asp Thr Lys Gly Arg Leu Tyr Arg Trp Arg Ser Pro Val
145 150 155
ATC ATA GAG AAA AGG GGC AAA GTT GAG GTC GAA GGT CAC CTG ATC GAC 950
Ile Ile Glu Lys Arg Gly Lys Val Glu Val Glu Gly His Leu Ile Asp
160 165 170 175
CTC AAA AGA GTT GTG CTT GAT GGT TCC GCG GCT ACC CCT GTA ACC AGA 998
Leu Lys Arg Val Val Leu Asp Gly Ser Ala Ala Thr Pro Val Thr Arg
180 185 190
GTT TCA GCG GAA CAA TGG AGT CGT CCT TAG ATGACTTCTG TCATGATAGC 1048
Val Ser Ala Glu Gln Trp Ser Arg Pro
195 200
ACGGCTCCAC AAAAGGTGCT CTTGGCGTTT TCTATTACCT ACACGCCAGT GATGATATAT 1108
GCCCTAAAGG TGAGTCGCGG CCGACTGCTA GGGCTTCTGC ACCTTTTGGT CTTCCTGAAT 1168
TGTGCTTTCA CCTTCGGGTA CATGACATTC GTGCACTTTC AGAGTACAAA TAAGGTCGCG 1228
CTCACTATGG GAGCAGTAGT TGCACTCCTT TGGGGGGTGT ACTCAGCCAT AGAAACCTGG 1288
AAATTCATCA CCTCCAGATG CCGTTTGTGC TTGCTAGGCC GCAAGTACAT TCTGGCCCCT 1348
GCCCACCACG TTGAAAGTGC CGCAGGCTTT CATCCGATTG CGGCAAATGA TAACCACGCA 1408
TTTGTCGTCC GGCGTCCCGG CTCCACTACG GTCAACGGCA CATTGGTGCC CGGGTTAAAA 1468
AGCCTCGTGT TGGGTGGCAG AAAAGCTGTT AAACAGGGAG TGGTAAACCT TGTTAAATAT 1528
GCCAAATAAC ACCGGCAAGC AGCAGAAGAG AAAGAAGGGG GATGGCCAGC CAGTCAATCA 1588
GCTGTGCCAG ATGCTGGGTA AGATCATCGC TCACCAAAAC CAGTCCAGAG GCAAGGGACC 1648
GGGAAAGAAA AATAAGAAGA AAAACCCGGA GAAGCCCCAT TTCCCTCTAG CGACTGAAGA 1708
TGATGTCAGA CATCACTTTA CCCCTAGTGA GCGTCAATTG TGTCTGTCGT CAATCCAGAC 1768
CGCCTTTAAT CAAGGCGCTG GGACTTGCAC CCTGTCAGAT TCAGGGAGGA TAAGTTACAC 1828
TGTGGAGTTT AGTTTGCCTA CGCATCATAC TGTGCGCCTG ATCCGCGTCA CAGCATCACC 1888
CTCAGCATGA TGGGCTGGCA TTCTTGAGGC ATCCCAGTGT TTGAATTGGA AGAATGCGTG 1948
GTGAATGGCA CTGATTGACA TTGTGCCTCT AAGTCACCTA TTCAATTAGG GCGACCGTGT 2008
47
CA 02418780 2003-02-28
GGGGGTAAGA TTTAATTGGC GAGAACCACA CGGCCGAAAT TA 2050
2) INFORMATION FOR SEQ ID N0:35:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH:200 amino acids
(B) TYPE: Amino Acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Protein
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Sus scrofa
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:35:
Met Leu Gly Lys Cys Leu Thr Ala Gly Cys Cys Ser Gln Leu Leu Phe
1 5 10 15
Leu Trp Cys Ile Val Pro Ser Cys Phe Val Ala Leu Val Ser Ala Asn
20 25 30
Gly Asn Ser Gly Ser Asn Leu Gln Leu Ile Tyr Asn Leu Thr Leu Cys
35 40 45
Glu Leu Asn Gly Thr Asp Trp Leu Ala Asn Lys Phe Asp Trp Ala Val
50 55 60
Glu Cys Phe Val Ile Phe Pro Val Leu Thr His Ile Val Ser Tyr Gly
65 70 75 80
Ala Leu Thr Thr Ser His Phe Leu Asp Thr Val Gly Leu Val Thr Val
85 90 95
Ser Thr Ala Gly Phe Val His Gly Arg Tyr Val Leu Ser Ser Met Tyr
100 105 110
Ala Val Cys Ala Leu Ala Ala Leu Ile Cys Phe Val Ile Arg Leu Ala
115 120 125
Lys Asn Cys Met Ser Trp Arg Tyr Ser Cys Thr Arg Tyr Thr Asn Phe
130 135 140
Leu Leu Asp Thr Lys Gly Arg Leu Tyr Arg Trp Arg Ser Pro Val Ile
145 150 155 160
Ile Glu Lys Arg Gly Lys Val Glu Val Glu Gly His Leu Ile Asp Leu
165 170 175
Lys Arg Val Val Leu Asp Gly Ser Ala Ala Thr Pro Val Thr Arg val
180 185 190
Ser Ala Glu Gln Trp Ser Arg Pro
195 200
(2) INFORMATION FOR SEQ ID N0:36:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 603 base pairs
(B) TYPE: Nucleic acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(1i) MOLECULE TYPE: DNA
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Sus scrofa
(ix) FEATURE:
(A) NAME/KEY:CDS
(B) LOCATION: (1)...(603)
(D) OTHER INFORMATION: wtROF5 of PRRSV strain 98-5579-1 GenBank
48
CA 02418780 2003-02-28
Accession #AF339500
(xij SEQUENCE DESCRIPTION: SEQ ID N0:36:
ATGTTGGGG AGATGCTTG ACCGCGGGCTAT TGCTCGCGA TTGCTTTCT 48
MetLeuGly ArgCysLeu ThrAlaGlyTyr CysSerArg LeuLeuSer
1 5 ZO 15
TTGTGGTGT ATCGTGCCG TTCTGGTTTGCT GTGCTCGTC AACGCCAAC 96
LeuTrpCys IleValPro PheTrpPheAla ValLeuVal AsnAlaAsn
20 25 30
AGCAACAGC AGCTCTCAT TTTCAGTTGATT TATAACTTG ACGCTATGC 144
SerAsnSex SerSerHis PheGlnLeuIle TyrAsnLeu ThrLeuCys
35 40 45
GAGCTGAAT GGCACAGAT TGGCTGGCCGAA AAATTTGAT TGGGCAGTG 192
GluLeuAsn GlyThrAsp TrpLeuAlaGlu LysPheAsp TrpAlaVal
50 55 60
GAGACTTTT GTCATCTTT CCCGTGTTGACT CACATTGTT TCCTATGGT 240
GluThrPhe ValIlePhe ProValLeuThr HisIleVal SerTyrGly
65 70 75 80
GCACTCACC ACCAGCCAT TTCCTTGATACA GTTGGTCTG GCTACTGTA 288
AlaLeuThr ThrSerHis PheLeuAspThr ValGlyLeu AlaThrVal
85 90 95
TCCACCGCC GGGTTTTAT CACAGGCGGTAT GTCTTGAGT AGCATCTAC 336
SerThrAla GlyPheTyr HisArgArgTyr ValLeuSer SerIleTyr
100 105 110
GCTGTCTGT GCTCTGGCT GCGTTGATTTGC TTCGTTATC AGGTTTgcg 384
AlaValCys AlaLeuAla AlaLeuIleCys PheValIle ArgPheAla
115 120 125
AAGAACTGC ATGTCCTGG CGCTACTCATGT ACCAGATAC ACCAACttc 432
LysAsnCys MetSerTrp ArgTyrSerCys ThrArgTyr ThrAsnPhe
130 135 140
CTTCTAGAT ACTAAGGGC AGACTCTATCGT TGGCGGTCG CCTGTCATT 480
LeuLeuAsp ThrLysGly ArgLeuTyrArg TrpArgSer ProValIle
145 150 155 160
ATAGAGAAA GGGGGTAAG GTTGAGGTCGAA GGCCACCTG ATCGACCTC 528
IleGluLys GlyGlyLys ValGluValGlu GlyHisLeu IleAspLeu
165 170 175
AAAAGAGTT GTGCTTGAT GGTTCCGTGGCA ACCCCTTTA ACCAGAGTT 576
LysArgVal ValLeuAsp GlySerValAla ThrProLeu ThrArgVal
180 185 190
TCAGCGGAA CAATGGTGT CGTCCCTAG 603
SerAlaGlu GlnTrpCys ArgPro
195 200
2)
INFORMATION
FOR
SEQ
ID
N0:37:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH:200 amino acids
(Bj TYPE: Amino Acid
49
CA 02418780 2003-02-28
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Protein
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Sus scrofa
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:37:
Met Leu Gly Arg Cys Leu Thr Ala Gly Tyr Cys Ser Arg Leu Leu Ser
1 5 10 15
Leu Trp Cys Ile Val Pro Phe Trp Phe Ala Val Leu Val Asn Ala Asn
20 25 30
Ser Asn Ser Ser Ser His Phe Gln Leu Ile Tyr Asn Leu Thr Leu Cys
35 40 45
Glu Leu Asn Gly Thr Asp Trp Leu Ala Glu Lys Phe Asp Trp Ala Val
50 55 60
Glu Thr Phe Val Ile Phe Pro Val Leu Thr His Ile Val Ser Tyr Gly
65 70 75 80
Ala Leu Thr Thr Ser His Phe Leu Asp Thr Val Gly Leu Ala Thr Val
85 90 95
Ser Thr Ala Gly Phe Tyr His Arg Arg Tyr Val Leu Ser Ser Ile Tyr
100 105 110
Ala Val Cys Ala Leu Ala Ala Leu Ile Cys Phe Val Ile Arg Phe Ala
115 120 125
Lys Asn Cys Met Ser Trp Arg Tyr Ser Cys Thr Arg Tyr Thr Asn Phe
130 135 140
Leu Leu Asp Thr Lys Gly Arg Leu Tyr Arg Trp Arg Ser Pro Val Ile
145 150 155 160
Ile Glu Lys Gly Gly Lys Val Glu Val Glu Gly His Leu Ile Asp Leu
165 170 175
Lys Arg Val Val Leu Asp Gly Ser Val Ala Thr Pro Leu Thr Arg Val
180 185 190
Ser Ala Glu Gln Trp Cys Arg Pro
195 200
(2) INFORMATION FOR SEQ ID N0:38:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 603 base pairs
(B) TYPE: Nucleic acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(ii) MOLECULE TYPE: DNA
(vi) ORIGINAL SOURCE:
(A) ORGANISM:Sus scrota
(ix) FEATURE:
(A) NAME/KEY:CDS
(B) LOCATION: (1)...(603)
(D) OTHER INFORMATION: wtORFS of PRRSV strain PRRSV57 GenBank
Accession #AF176477
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:38:
ATG TTG GAG AAA TGC TTG ACC GCG GGC TGT TGC TCG CGA TTG CTT TCT 48
Met Leu Glu Lys Cys Leu Thr Ala Gly Cys Cys Ser Arg Leu Leu Ser
1 5 10 15
$~
CA 02418780 2003-02-28
TTG TGG TGT ATC GTG CCG TTC TGT TTT GCT GCG CTC GCC AAC GCC AGC 96
Leu Trp Cys Ile Val Pro Phe Cys Phe Ala Ala Leu Ala Asn Ala Ser
20 25 30
AAC AAC AGC AGC TCC CAT CTA CAG CTG ATT TAC AAC TTG ACG CTA TGT 144
Asn Asn Ser Ser Ser His Leu Gln Leu Ile Tyr Asn Leu Thr Leu Cys
35 40 45
GAG CTG AAT GGC ACA GAT TGG CTA GCT GAC AAT TTT GAT TGG GCA GTG 192
Glu Leu Asn Gly Thr Asp Trp Leu Ala Asp Asn Phe Asp Trp Ala Val
50 55 60
GAG AGT TTT GTC ATC TTT CCC GTT TTG ACT CAC ATT GTC TCC TAT GGT 240
Glu Ser Phe Val Ile Phe Pro Val Leu Thr His Ile Val Ser Tyr GIy
65 70 75 80
GCC CTC ACT ACC AGC CAT TTC CTT GAC ACA GTC GCT TTA GTC ACT GTG 288
Ala Leu Thr Thr Ser His Phe Leu Asp Thr Val Ala Leu Val Thr Val
85 90 95
TCT ACC GCC GGG TTT GTT CAC GGG CGG TAT GTC CTA AGT AGC ATC TAC 336
Ser Thr Ala Gly Phe Val His Gly Arg Tyr Val Leu Ser Ser Ile Tyr
100 105 110
GCG GTC TGT GCC CTG GCT GCG TTG ACT TGC TTC GTC ATT AGG TTT GCA 384
Ala Val Cys Ala Leu Ala Ala Leu Thr Cys Phe Val Ile Arg Phe Ala
115 120 125
AAG AAT TGC ATG TCC TGG CGC TAC GCG TGT ACC AGA TAT ACC AAC TTT 432
Lys Asn Cys Met Ser Trp Arg Tyr Ala Cys Thr Arg Tyr Thr Asn Phe
130 135 140
CTT CTG GAC ACT AAG GGC AGA CTC TAT CGT TGG CGG TCG CCT GTC ATC 480
Leu Leu Asp Thr Lys Gly Arg Leu Tyr Arg Trp Arg Ser Pro Val Ile
145 150 155 160
ATA GAG AAA AGG GGC AAA GTT GAG GTC GAA GGT CAC CTG ATC GAC CTC 528
Ile G1u Lys Arg Gly Lys Val Glu Val Glu Gly His Leu Ile Asp Leu
165 170 175
AAA AGA GTT GTG CTT GAT GGT TCC GTG GCA ACC CCT ATA ACC AGA GTT 576
Lys Arg Val Val Leu Asp Gly Ser Val Ala Thr Pro Ile Thr Arg Val
180 185 190
TCA GCG GAA CAA TGG GAT CGT CCT TAG 603
Ser Ala Glu Gln Trp Asp Arg Pro
195 200
2) INFORMATION FOR SEQ ID N0:39:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH:200 amino acids
(B) TYPE: Amino Acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Protein
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Sus scrofa
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:39:
51
CA 02418780 2003-02-28
Met Leu Glu Lys Cys Leu Thr Ala Gly Cys Cys Ser Arg Leu Leu Ser
1 5 10 15
Leu Trp Cys Ile Val Pro Phe Cys Phe Ala Ala Leu Ala Asn Ala Ser
20 25 30
Asn Asn Ser Ser Ser His Leu Gln Leu Ile Tyr Asn Leu Thr Leu Cys
35 40 45
Glu Leu Asn Gly Thr Asp Trp Leu Ala Asp Asn Phe Asp Trp Ala Val
50 55 60
Glu Ser Phe Val Ile Phe Pro Val Leu Thr His Ile Val Ser Tyr Gly
65 70 75 80
Ala Leu Thr Thr Ser His Phe Leu Asp Thr Val Ala Leu Val Thr Val
85 90 95
Ser Thr Ala Gly Phe Val His Gly Arg Tyr Val Leu Ser Ser Ile Tyr
100 105 110
Ala Val Cys Ala Leu Ala Ala Leu Thr Cys Phe Val Ile Arg Phe Ala
115 120 125
Lys Asn Cys Met Ser Trp Arg Tyr Ala Cys Thr Arg Tyr Thr Asn Phe
130 135 140
Leu Leu Asp Thr Lys Gly Arg Leu Tyr Arg Trp Arg Ser Pro Val Ile
145 150 155 160
Ile Glu Lys Arg Gly Lys Val Glu Val Glu Gly His Leu Ile Asp Leu
165 170 175
Lys Arg Val Val Leu Asp Gly Ser Val Ala Thr Pro Ile Thr Arg Val
180 185 190
Ser Ala Glu Gln Trp Asp Arg Pro
195 200
(2) INFORMATION FOR SEQ ID N0:40:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 603 base pairs
(B) TYPE: Nucleic acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(ii) MOLECULE TYPE: DNA
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Sus scrofa
(ix) FEATURE:
(A) NAME/KEY:CDS
(B) LOCATION: (1)...(603)
(D) OTHER INFORMATION: synORF5 variant of IAF-Klop (Figure 8)
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:40:
ATG CTG GGC AAG TGC CTG ACC GCC GGC TGT TGC TCA CAG CTG CCC TTC 48
Met Leu Gly Lys Cys Leu Thr Ala Gly Cys Cys Ser Gln Leu Pro Phe
1 5 10 15
CTG TGG TGT ATC GTG CCC TTC TGT TTC GCC GCC CTG GTG AAC GCC TCC 96
Leu Trp Cys Ile Val Pro Phe Cys Phe Ala Ala Leu Val Asn Ala Ser
20 25 30
TCG TCC TCG TCG TCC CAG CTG CAG TCC ATC TAC AAC CTG ACC ATC TAT 144
Ser Ser Ser Ser Ser Gln Leu Gln Ser Ile Tyr Asn Leu Thr Ile Tyr
35 40 45
52
CA 02418780 2003-02-28
GAGCTG GGC ACCGACTGG CTG AAG TTCGACTGG TCCGTG 192
AAC AAC AAC
GluLeuAsnGly ThrAspTrp LeuAsnLys AsnPheAspTrp SerVal
50 55 60
GAGACCTTCGTG ATCTTCCCC GTGCTGACC CACATCGTGTCC TACGGC 240
GluThrPheVal IlePhePro ValLeuThr HisIleValSer TyrGly
65 70 75 80
GCCCTGACCACC TCCCACTTC CTGGACGCC GTGGGCCTGATC ACCGTG 288
AlaLeuThrThr SerHisPhe LeuAspAla ValGlyLeuIle ThrVal
85 90 95
TCCACCGCCGGC TACTACCAC GGCCGCTAC GTACTGTCCTCC GTGTAC 336
SerThrAlaGly TyrTyrHis GlyArgTyr ValLeuSerSer ValTyr
100 105 110
GCCGTGTGCGCC CTGGCCGCC CTGATCTGC TTCGTGATCCGC CTGACA 384
AlaValCysAla LeuAlaAla LeuIleCys PheValIleArg LeuThr
115 120 125
AAGAACTGCATG TCCTGGCGC TACTCCTGT ACACGCTACACC AACTTC 432
LysAsnCysMet SerTrpArg TyrSerCys ThrArgTyrThr AsnPhe
130 135 140
CTGCTGGACTCC AAGGGCAAG CTGTATCGC TTGCGCTCCCCC GTGATC 480
LeuLeuAspSer LysGlyLys LeuTyrArg LeuArgSerPro ValIle
145 150 155 160
ATCGAGAAGGGC GGCAAGGTG GAGGTGGAC GGCCACCTGATC GACCTG 528
IleGluLysGly GlyLysVal GluValAsp GlyHisLeuIle AspLeu
165 170 175
AAGCGCGTGGTG CTGGACGCT TCCGCCGCC ACCCCCGTGACC AAGGTG 576
LysArgValVal LeuAspAla SerAlaAla ThrProValThr LysVal
180 185 190
TCCGCGGAGCAG TGGTGTCGC CCCTAG 603
SerAlaGluGln TrpCysArg Pro
195 200
(2) INFORMATION FOR SEQ ID N0:41:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 200 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY:
(ii) MOLECULE TYPE: Protein
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Sus scrofa
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:41:
Met Leu Gly Lys Cys Leu Thr Ala Gly Cys Cys Ser Gln Leu Pro Phe
1 5 10 15
Leu Trp Cys Ile Val Pro Phe Cys Phe Ala Ala Leu Val Asn Ala Ser
20 25 30
53
CA 02418780 2003-02-28
Ser Ser Ser Ser Ser Gln Leu Gln Ser Ile Tyr Asn Leu Thr Ile Tyr
35 40 45
Glu Leu Asn Gly Thr Asp Trp Leu Asn Lys Asn Phe Asp Trp Ser Val
50 55 60
Glu Thr Phe Val Ile Phe Pro Val Leu Thr His Ile Val Ser Tyr Gly
65 70 75 80
Ala Leu Thr Thr Ser His Phe Leu Asp Ala Val Gly Leu Ile Thr Val
85 90 95
Ser Thr Ala Gly Tyr Tyr His Gly Arg Tyr Val Leu Ser Ser Val Tyr
100 105 110
Ala Val Cys Ala Leu Ala Ala Leu Ile Cys Phe Val Ile Arg Leu Thr
115 120 125
Lys Asn Cys Met Ser Trp Arg Tyr Ser Cys Thr Arg Tyr Thr Asn Phe
130 135 140
Leu Leu Asp Ser Lys Gly Lys Leu Tyr Arg Leu Arg Ser Pro Val Ile
145 150 155 160
Ile Glu Lys Gly Gly Lys Val Glu Val Asp Gly His Leu Ile Asp Leu
165 170 175
Lys Arg Val Val Leu Asp Ala Ser Ala Ala Thr Pro Val Thr Lys Val
180 185 190
Ser Ala Glu Gln Trp Cys Arg Pro
195 200
(2) INFORMATION FOR SEQ ID N0:42:
(i)SEQUENCE CHARACTERISTICS:
(A) LENGTH: 537
base pairs
(B) TYPE: Nucleicacid
(C) STRANDEDNESS:Single
(D) TOPOLOGY:
Linear
(ii)MOLECULE TYPE:
DNA
(vi)ORIGINAL SOURCE:
(A) ORGANISM: scrofa
Sus
(ix)FEATURE:
(A) NAME/KEY:CDS
(B) LOCATION: ...(537)
(1)
(D) OTHER INFORMATION: of IAF-Klop (GenBank
wtORF4 #AF003345)
(xi)SEQUENCE
DESCRIPTION:
SEQ
ID
N0:42:
ATGGCTGCG TCC CTT CTT CTC GTT GGTTTTGAACGT CTCTTG 48
TTC TTG
MetAlaAla Ser Leu Leu Leu Val GlyPheGluArg LeuLeu
Phe Leu
1 5 10 15
GTTTCTCAG GCG TTC GCC AAA TGT TTCAGTTCGAGT CTTTCA 96
TGC CCA
ValSerGln Ala Phe Ala Lys Cys PheSerSerSer LeuSer
Cys Pro
20 25 30
GACATCGAG ACC AAC ACC ACA GCG AGTTCTGTCGTC CTCCAA 144
ACC GCA
AspIleGlu Thr Asn Thr Thr Ala SerSerValVal LeuGln
Thr Ala
35 40 45
GACATTAGC TGT CTT AGG GGC TCG TCCTCTGAGACG ATTCGC 192
CAT TAC
AspIleSer Cys Leu Arg Gly Ser SerSerGluThr IleArg
His Tyr
50 55 60
AAAATCCCT CAG TGC CGC GCG GGG ACACCCGTGTAC ATCACT 240
ACG ATA
LysIlePro Gln Cys Arg Ala Gly ThrProValTyr IleThr
Thr Ile
54
CA 02418780 2003-02-28
65 70 75 80
ATC ACG GCC AAC GTA ACA GAT GAG AAT TAT TTG CAT TCC TCT GAC CTC 288
Ile Thr Ala Asn Val Thr Asp Glu Asn Tyr Leu His Ser Ser Asp Leu
85 90 95
CTCATGCTT TCTTCTTGCCTT TTCTATGCT TCTGAGATG AGTGAAAAG 336
LeuMetLeu SerSerCysLeu PheTyrAla SexGluMet SerGluLys
100 105 110
GGATTTAAG GTGATATTTGGC AATGTGTCA GGCATCGTT TCTGTGTGT 384
GlyPheLys ValIlePheGly AsnValSer GlyIleVal SerValCys
115 120 125
GTTAATTTT ACCAGCTATGTC CAACACGTC AAGGAGTTC ACCCAACGC 432
ValAsnPhe ThrSerTyrVal GlnHisVal LysGluPhe ThrGlnArg
130 135 140
TCCTTAATA GTCGACCATGTG CGACTGCTC CATTTCATG ACACCTGAG 480
SerLeuIle ValAspHisVal ArgLeuLeu HisPheMet ThrProGlu
145 150 155 160
ACCATGAGG TGGGCAACCGTT TTAGCCTGT CTTTTCGCC ATTCTGTTG 528
ThrMetArg TrpAlaThrVal LeuAlaCys LeuPheAla IleLeuLeu
165 170 175
GCAATTTGA 537
AlaIIe
(2) INFORMATION FOR SEQ ID N0:43:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 178 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY:
(ii) MOLECULE TYPE: Protien
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Sus scrofa
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:4~:
Met Ala Ala Ser Leu Leu Phe Leu Leu Val Gly Phe Glu Arg Leu Leu
1 5 10 15
Val Ser GIn Ala Phe AIa Cys Lys Pro Cys Phe Ser Ser Ser Leu Ser
20 25 30
Asp Ile Glu Thr Asn Thr Thr Thr Ala Ala Ser Ser Val Val Leu Gln
35 40 45
Asp Ile Ser Cys Leu Arg His Gly Tyr Ser Ser Ser Glu Thr Ile Arg
50 55 60
Lys Ile Pro Gln Cys Arg Thr Ala Ile Gly Thr Pro Val Tyr Ile Thr
65 70 75 BO
Ile Thr Ala Asn Val Thr Asp Glu Asn Tyr Leu His Ser Ser Asp Leu
85 90 95
Leu Met Leu Ser Ser Cys Leu Phe Tyr Ala Ser Glu Met Ser Glu Lys
100 105 110
Gly Phe Lys Val Ile Phe Gly Asn Val Ser Gly Ile Val Ser Val Cys
115 120 125
Val Asn Phe Thr Ser Tyr Val Gln His Val Lys Glu Phe Thr Gln Arg
CA 02418780 2003-02-28
130 135 140
Ser Leu Ile Val Asp His Val Arg Leu Leu His Phe Met Thr Pro Glu
145 150 155 160
Thr Met Arg Trp Ala Thr Val Leu Ala Cys Leu Phe Ala Ile Leu Leu
165 170 175
Ala Ile
(2) INFORMATION FOR SEQ ID N0:44:
(i)SEQUENCE CHARACTERISTICS:
(A) LENGTH: 37 pairs
5 base
(B) TYPE: leicaci d
Nuc
(C) STRANDEDNESS: Sin gle
(D) TOPOLOGY:Linear
(ii)MOLECULE TYPE:DNA
(vi)ORIGINAL SOURCE:
(A) ORGANISM:Susscrofa
(ix)FEATURE:
(A) NAME/KEY:CDS
(B) LOCATION:(1)...(537)
(D) OTHER : IAF-Klop
INFORMATION synORF4
of
(xi)SEQUENCE ID :
DESCRIPTION: N0:44
SEQ
ATGGCCGCC TCC CTG TTCCTG CTGGTGGGC TTTGAGCGC CTGCTG 48
CTG
MetAlaAla Ser Leu PheLeu LeuValGly PheGluArg LeuLeu
Leu
1 5 10 15
GTGTCCCAG GCC TTC TGCAAG CCCTGCTTC TCCTCCTCC CTGTCC 96
GCC
ValSerGln Ala Phe CysLys ProCysPhe SerSerSer LeuSer
Ala
20 25 30
GACATCGAG ACC AAC ACCACC GCCGCCTCC TCCGTGGTG CTGCAG 144
ACC
AspIleGlu Thr Asn ThrThr AlaAlaSer SerValVal LeuGln
Thr
35 40 45
GACATCTCC TGC CTG CACGGC TACTCCTCC TCCGAGACC ATCCGC 192
CGC
AspIleSer Cys Leu HisGly TyrSerSer SerGluThr IleArg
Arg
50 55 60
AAGATCCCC CAG TGC ACCGCC ATCGGCACC CCCGTGTAC ATCACC 240
CGC
LysIlePro Gln Cys ThrAla IleGlyThr ProValTyr IleThr
Arg
65 70 75 BO
ATCACCGCC AAC GTG GACGAG AACTACCTG CACTCCTCC GACCTG 288
ACC
IleThrAla Asn Val AspGlu AsnTyrLeu HisSerSer AspLeu
Thr
85 90 95
CTGATGCTG TCC TCC CTGTTC TACGCCTCC GAGATGTCC GAGAAG 336
TGC
LeuMetLeu Ser Ser LeuPhe TyrAlaSer GluMetSer GluLys
Cys
100 105 110
GGCTTTAAG GTG ATC GGCAAC GTGTCCGGC ATCGTGTCC GTGTGC 384
TTT
GlyPheLys Val Ile GlyAsn ValSerGly IleValSer ValCys
Phe
115 120 125
GTGAACTTT ACC TCC GTGCAG CACGTGAAG GAGTTCACC CAGCGC 432
TAC
56
CA 02418780 2003-02-28
Val Asn Phe Thr Ser Tyr Val Gln His Val Lys Glu Phe Thr Gln Arg
130 135 140
TCC CTG ATC GTG GAC CAC GTG CGC CTG CTG CAC TTC ATG ACC CCC GAG 480
Ser Leu Ile Val Asp His Val Arg Leu Leu His Phe Met Thr Pro Glu
145 150 155 160
ACC ATG CGC TGG GCC ACC GTG CTG GCC TGC CTG TTC GCC ATC CTG CTG 528
Thr Met Arg Trp Ala Thr Val Leu Ala Cys Leu Phe Ala Ile Leu Leu
165 170 175
GCC ATC TGA 537
Ala Ile
(2) INFORMATION FOR SEQ ID N0:45:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 178 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(ii) MOLECULE TYPE: Protein
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Sus scrofa
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:45:
Met Ala Ala Ser Leu Leu Phe Leu Leu Val Gly Phe Glu Arg Leu Leu
1 5 10 15
Val Ser Gln Ala Phe Ala Cys Lys Pro Cys Phe Ser Ser Ser Leu Ser
20 25 30
Asp Ile Glu Thr Asn Thr Thr Thr Ala Ala Ser Ser Val Val Leu Gln
35 40 45
Asp Ile Ser Cys Leu Arg His Gly Tyr Ser Ser Ser Glu Thr Ile Arg
50 55 60
Lys Ile Pro Gln Cys Arg Thr Ala Ile Gly Thr Pro Val Tyr Ile Thr
65 70 75 80
Ile Thr Ala Asn Val Thr Asp Glu Asn Tyr Leu His Ser Ser Asp Leu
85 90 95
Leu Met Leu Ser Ser Cys Leu Phe Tyr Ala Ser Glu Met Ser Glu Lys
100 105 110
Gly Phe Lys Val Ile Phe Gly Asn Val Ser Gly Ile Val Ser Val Cys
115 120 125
Val Asn Phe Thr Ser Tyr Val Gln His Val Lys Glu Phe Thr Gln Arg
130 135 140
Ser Leu Ile Val Asp His Val Arg Leu Leu His Phe Met Thr Pro Glu
145 150 155 160
Thr Met Arg Trp Ala Thr Val Leu Ala Cys Leu Phe Ala Ile Leu Leu
165 170 175
Ala Ile
(2) INFORMATION FOR SEQ ID N0:46:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 525 base pairs
(B) TYPE: Nucleic acid
57
CA 02418780 2003-02-28
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(ii)MOLECULE DNA
TYPE:
(vi)ORIGINAL
SOURCE:
(A) ORGANISM: Susscrofa
(ix)FEATURE:
(A) NAME/KEY:CDS
(B) LOCATION: (1)...(525)
(D) OTHERINFORMATION : of IAF-Klop(GenBank
wtORF6 #
U64928)
(xi)SEQUENCE ID
DESCRIPTION: N0:46:
SEQ
ATGGTG TCG TCC GAC GACTTCTGCAAT GACAGCACG GCCCCACAA 48
CTA
MetVal Ser Ser Asp AspPheCysAsn AspSerThr AlaProGln
Leu
1 5 10 15
AAGGTA CTC CTG TTT TCTATCACCTAC ACGCCAGTA ATGATATAT 96
GCG
LysVal Leu Leu Phe SerIleThrTyr ThrProVal MetIleTyr
Ala
20 25 30
GCCCTA AAG GTA CGC GGCCGACTGCTA GGGCTTCTG CACCTTTTA 144
AGT
AlaLeu Lys Val Arg GlyArgLeuLeu GlyLeuLeu HisLeuLeu
Ser
35 40 45
ATTTTC CTG AAT GCT TTCACCTTCGGG TATATGACA TTCGCGCAC 192
TGT
IlePhe Leu Asn Ala PheThrPheGly TyrMetThr PheAlaHis
Cys
50 55 60
TTTCAG AGT ACA AAA GTCGCGCTCACT ATGGGAGCA GTAGTTGCG 240
AAT
PheGln Ser Thr Lys ValAlaLeuThr MetGlyAla ValValAla
Asn
65 70 75 $0
CTCCTT TGG GGG TAC TCAGCCATAGAA ACCTGGAAA TTCATCACC 288
GTG
LeuLeu Trp Gly Tyr SerAlaIleGlu ThrTrpLys PheIleThr
Val
85 90 95
TCCAGA TGC CGT TGC TTGCTAGGCCGC AAGTACATT CTGGCCCCT 336
TTG
SerArg Cys Arg Cys LeuLeuGlyArg LysTyrIle LeuAlaPro
Leu
100 105 110
GCCCAC CAC GTT AGT GCCGCAGGCTTT CATCCGATT GCGGCAAGT 384
GAG
AlaHis His Val Ser AlaAlaGlyPhe HisProIle AlaAlaSer
Glu
115 120 125
GATAAC CAC GCA GTC GTCCGGCGTCCC GGCTCCACT ACGGTTAAC 432
TTT
AspAsn His Ala Val ValArgArgPro GlySexThr ThrValAsn
Phe
130 135 140
GGCACA TTG GTG GGG TTGAAAAGCCTC GTGTTGGGT GGCAGAAAA 480
CCC
GlyThr Leu Val Gly LeuLysSerLeu ValLeuGly GlyArgLys
Pro
145 150 155 160
GCTGTC AAA CGG GTG GTAAACCTCGTT AAATATGCC AAATAA 525
GGA
AlaVal Lys Arg Val ValAsnLeuVal LysTyrAla Lys
Gly
165 170
S$
CA 02418780 2003-02-28
(2) INFORMATION FOR SEQ ID N0:47:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 174 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(ii) MOLECULE TYPE: DNA
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Sus scrofa
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:47:
Met Val Ser Ser Leu Asp Asp Phe Cys Asn Asp Ser Thr Ala Pro Gln
1 5 10 15
Lys Val Leu Leu Ala Phe Ser Ile Thr Tyr Thr Pro Val Met Ile Tyr
20 25 30
Ala Leu Lys Val Ser Arg Gly Arg Leu Leu Gly Leu Leu His Leu Leu
35 40 45
Ile Phe Leu Asn Cys Ala Phe Thr Phe Gly Tyr Met Thr Phe Ala His
50 55 60
Phe Gln Ser Thr Asn Lys Val Ala Leu Thr Met Gly Ala Val Val Ala
65 70 75 80
Leu Leu Trp Gly Val Tyr Ser Ala Ile Glu Thr Trp Lys Phe Ile Thr
85 90 95
Ser Arg Cys Arg Leu Cys Leu Leu Gly Arg Lys Tyr Ile Leu Ala Pro
100 105 110
Ala His His Val Glu Ser Ala Ala Gly Phe His Pro Ile Ala Ala Ser
115 120 125
Asp Asn His Ala Phe Val Val Arg Arg Pro Gly Ser Thr Thr Val Asn
130 135 140
Gly Thr Leu Val Pro Gly Leu Lys Ser Leu Val Leu Gly Gly Arg Lys
145 150 155 160
Ala Val Lys Arg Gly Val Val Asn Leu Val Lys Tyr Ala Lys
165 170
(2) INFORMATION FOR SEQ ID N0:48:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 525 base pairs
(B) TYPE: Nucleic acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(ii) MOLECULE TYPE: DNA
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Sus scrofa
(ix) FEATURE:
(A) NAME/KEY:CDS
(B) LOCATION: (1)...(525)
(D) OTHER INFORMATION: synORF6 of IAF-Klop
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:48:
ATG GTG TCC TCC CTG GAC GAC TTC TGC AAC GAC TCC ACC GCC CCC CAG 48
Met Val Ser Ser Leu Asp Asp Phe Cys Asn Asp Ser Thr Ala Pro Gln
59
CA 02418780 2003-02-28
1 5 10 15
AAGGTGCTG CTGGCCTTCTCC ATCACCTAC ACCCCCGTGATG ATCTAC 96
LysValLeu LeuAlaPheSer IleThrTyr ThrProValMet IleTyr
20 25 30
GCCCTGAAG GTGTCCCGCGGC CGCCTGCTG GGCCTGCTGCAC CTGCTG 144
AlaLeuLys ValSerArgGly ArgLeuLeu GlyLeuLeuHis LeuLeu
35 40 45
ATCTTCCTG AACTGCGCCTTC ACCTTCGGC TACATGACCTTC GCCCAC 192
IlePheLeu AsnCysAlaPhe ThrPheGly TyrMetThrPhe AlaHis
50 55 60
TTCCAGTCC ACCAACAAGGTG GCCCTGACC ATGGGCGCCGTG GTGGCC 240
PheGlnSer ThrAsnLysVal AlaLeuThr MetGlyAlaVal ValAla
65 70 75 80
CTGCTGTGG GGCGTGTACTCC GCCATCGAG ACCTGGAAGTTC ATCACC 288
LeuLeuTrp GlyValTyrSer AlaIleGlu ThrTrpLysPhe IleThr
85 90 95
TCCCGCTGC CGCCTGTGCCTG CTGGGCCGC AAGTACATCCTG GCCCCC 336
SerArgCys ArgLeuCysLeu LeuGlyArg LysTyrIleLeu AlaPro
100 105 110
GCCCACCAC GTGGAGTCCGCC GCCGGCTTC CACCCCATCGCC GCCTCC 384
AlaHisHis ValGluSerAla AlaGlyPhe HisProIleAla AlaSer
115 120 125
GACAACCAC GCCTTCGTGGTG CGCCGCCCC GGCTCCACCACC GTGAAC 432
AspAsnHis AlaPheValVal ArgArgPro GlySerThrThr ValAsn
130 135 140
GGCACCCTG GTGCCCGGCCTG AAGTCCCTG GTGCTGGGCGGC CGCAAG 480
GlyThrLeu ValProGlyLeu LysSerLeu ValLeuGlyGly ArgLys
145 150 155 160
GCCGTGAAG CGCGGCGTGGTG AACCTGGTG AAGTACGCCAAG TAA 525
AlaValLys ArgGlyValVal AsnLeuVal.LysTyrAlaLys
165 170
(2)INFORMATION FORSEQID
N0:49:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 174 Amino acid
(B) TYPE: amino acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY:
(ii) MOLECULE TYPE: Protein
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Sus scrofa
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:49:
Met Val Ser Ser Leu Asp Asp Phe Cys Asn Asp Ser Thr Ala Pro Gln
1 5 10 15
Lys Val Leu Leu Ala Phe Ser Ile Thr Tyr Thr Pro Val Met Ile Tyr
CA 02418780 2003-02-28
20 25 30
Ala Leu Lys Val Ser Arg Gly Arg Leu Leu Gly Leu Leu His Leu Leu
35 40 45
Ile Phe Leu Asn Cys Ala Phe Thr Phe Gly Tyr Met Thr Phe Ala His
50 55 60
Phe Gln Ser Thr Asn Lys Val Ala Leu Thr Met Gly Ala Val Val Ala
65 70 75 80
Leu Leu Trp Gly Val Tyr Ser Ala Ile Glu Thr Trp Lys Phe Ile Thr
85 90 95
Ser Arg Cys Arg Leu Cys Leu Leu Gly Arg Lys Tyr Tle Leu Ala Pro
100 105 110
Ala His His Val Glu Ser Ala Ala Gly Phe His Pro Ile Ala Ala Ser
115 120 125
Asp Asn His Ala Phe Val Val Arg Arg Pro Gly Ser Thr Thr Val Asn
130 135 140
Gly Thr Leu Val Pro Gly Leu Lys Ser Leu Val Leu Gly Gly Arg Lys
145 I50 155 160
Ala Val Lys Arg Gly Val Val Asn Leu Val Lys Tyr Ala Lys
165 170
(2) INFORMATION FOR SEQ ID N0:50:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 69 base pairs
(B) TYPE: Nucleic acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(ii) MOLECULE TYPE: DNA
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Artificial sequence
(ix) FEATURE:
(D) OTHER INFORMATION: oligonucleotide primer 1
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:50:
ACCGGATCCA TGCTGGGCAA GTGCCTGACC GCCGGCTGTT GCTCCCAGCT GCCCTTCCTG 60
TGGTGTATC 69
(2) INFORMATION FOR SEQ ID N0:51:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 60 base pairs
(B) TYPE: Nucleic acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(ii) MOLECULE TYPE: DNA
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Artificial sequence
(ix) FEATURE:
(D) OTHER INFORMATION: oligonucleotide primer 2
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:51:
61
CA 02418780 2003-02-28
GTGCCCTTCT GTTTCGCCGC CCTGGTGAAC GCCTCCTCCT CCTCCTCCTC CCAGCTGCAG 60
(2) INFORMATION FOR SEQ ID N0:52:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 60 base pairs
(B) TYPE: Nucleic acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(ii) MOLECULE TYPE: DNA
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Artificial sequence
(ix) FEATURE:
(D) OTHER INFORMATION: oligonucleotide primer 3
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:52:
TCCATCTACA ACCTGACCAT CTGTGAGCTG AACGGCACCG ACTGGCTGAA CAAGAACTTC 60
(2) INFORMATION FOR SEQ ID N0:53:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 60 base pairs
(B) TYPE: Nucleic acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(ii) MOLECULE TYPE: DNA
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Artificial sequence
(ix) FEATURE:
(D) OTHER INFORMATION: oligonucleotide primer 4
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:53:
GACTGGGCCG TGGAGACCTT CGTGATCTTC CCCGTGCTGA CCCACATCGT GTCCTACGGC 60
(2) INFORMATION FOR SEQ ID N0:54:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 49 base pairs
(B) TYPE: Nucleic acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(ii) MOLECULE TYPE: DNA
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Artificial sequence
(ix) FEATURE:
(D) OTHER INFORMATION: oligonucleotide primer 5
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:54:
G2
CA 02418780 2003-02-28
GCCCTGACCA CCTCCCACTT CCTGGACGCC GTGGGCCTGA TCACCGTGT 49
(2) INFORMATION FOR SEQ ID N0:55:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 60 base pairs
(B) TYPE: Nucleic acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(ii) MOLECULE TYPE: DNA
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Artificial sequence
(ix) FEATURE:
(D) OTHER INFORMATION: oligonucleotide primer 6
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:55:
TACTACCACG GCCGCTACGT GCTGTCCTCC GTGTACGCCG TGTGCGCCCT GGCCGCCCTG 60
(2) INFORMATION FOR SEQ ID N0:56:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 60 base pairs
(B) TYPE: Nucleic acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(ii) MOLECULE TYPE: DNA
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Artificial sequence
(ix) FEATURE:
(D) OTHER INFORMATION: oligonucleotide primer 7
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:56:
ATCTGCTTCG TGATCCGCCT GACCAAGAAC TGCATGTCCT GGCGCTACTC CTGTACCCGC 60
(2) INFORMATION FOR SEQ ID N0:57:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 60 base pairs
(B) TYPE: Nucleic acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(ii) MOLECULE TYPE: DNA
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Artificial sequence
(ix) FEATURE:
(D) OTHER INFORMATION: oligonucleotide primer 8
63
CA 02418780 2003-02-28
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:57:
TACACCAACT TCCTGCTGGA CTCCAAGGGC AAGCTGTACC GCTGGCGCTC CCCCGTGATC 60
(2) INFORMATION FOR SEQ ID N0:58:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 60 base pairs
(B) TYPE: Nucleic acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(ii) MOLECULE TYPE: DNA
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Artificial sequence
(ix) FEATURE:
(D) OTHER INFORMATION: oligonucleotide primer 9
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:58:
ATCGAGAAGG GCGGCAAGGT GGAGGTGGAC GGCCACCTGA TCGACCTGAA GCGCGTGGTG 60
(2) INFORMATION FOR SEQ ID N0:59:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 60 base pairs
(B) TYPE: Nucleic acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(ii) MOLECULE TYPE: DNA
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Artificial sequence
(ix) FEATURE:
(D) OTHER INFORMATION: oligonucleotide primer 10
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:59:
CTGGACGGCT CCGCCGCCAC CCCCGTGACC AAGGTGTCCG CCGAGCAGTG GTGTCGCCCC 60
(2) INFORMATION FOR SEQ ID N0:60:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: Nucleic acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(ii) MOLECULE TYPE: DNA
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Artificial sequence
(ix) FEATURE:
(D) OTHER INFORMATION: oligonucleotide primer 11RC
64
CA 02418780 2003-02-28
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:60:
ACAGCCGGCG GTCAGGCACT TGCCCAGCAT 30
(2) INFORMATION FOR SEQ ID N0:61:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 60 base pairs
(B) TYPE: Nucleic acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(ii) MOLECULE TYPE: DNA
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Artificial sequence
(ix) FEATURE:
(D) OTHER INFORMATION: oligonucleotide primer 1RC
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:61:
GTTCACCAGG GCGGCGAAAC AGAAGGGCAC GATACACCAC AGGAAGGGCA GCTGGGAGCA 60
(2) INFORMATION FOR SEQ ID N0:62:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 60 base pairs
(B) TYPE: Nucleic acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(ii) MOLECULE TYPE: DNA
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Artificial sequence
(ix) FEATURE:
(D) OTHER INFORMATION: oligonucleotide primer 2RC
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:62:
CAGCTCACAG ATGGTCAGGT TGTAGATGGA CTGCAGCTGG GAGGAGGAGG AGGAGGAGGC 60
(2) INFORMATION FOR SEQ ID N0:63:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 60 base pairs
(B) TYPE: Nucleic acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(ii) MOLECULE TYPE: DNA
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Artificial sequence
(ix) FEATURE:
CA 02418780 2003-02-28
(D) OTHER INFORMATION: oligonucleotide primer 3RC
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:63:
GAAGATCACG AAGGTCTCCA CGGCCCAGTC GAAGTTCTTG TTCAGCCAGT CGGTGCCGTT 60
(2) INFORMATION FOR SEQ ID N0:64:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 60 base pairs
(B) TYPE: Nucleic acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(ii) MOLECULE TYPE: DNA
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Artificial sequence
(ix) FEATURE:
(D) OTHER INFORMATION: oligonucleotide primer 4RC
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:64:
GGCGTCCAGG AAGTGGGAGG TGGTCAGGGC GCCGTAGGAC ACGATGTGGG TCAGCACGGG 60
(2) INFORMATION FOR SEQ ID N0:65:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 60 base pairs
(B) TYPE: Nucleic acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(ii) MOLECULE TYPE: DNA
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Artificial sequence
(ix) FEATURE:
(D) OTHER INFORMATION: oligonucleotide primer 5RC
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:65:
GGAGGACAGC ACGTAGCGGC CGTGGTAGTA GCCGGCGGTG GACACGGTGA TCAGGCCCAC 60
(2) INFORMATION FOR SEQ ID N0:66:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 60 base pairs
(B) TYPE: Nucleic acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(ii) MOLECULE TYPE: DNA
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Artificial sequence
66
CA 02418780 2003-02-28
(ix) FEATURE:
(D) OTHER INFORMATION: oligonucleotide primer 6RC
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:66:
GTTCTTGGTC AGGCGGATCA CGAAGCAGAT CAGGGCGGCC AGGGCGCACA CGGCGTACAC 60
(2) INFORMATION FOR SEQ ID N0:67:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 60 base pairs
(B) TYPE: Nucleic acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(ii) MOLECULE TYPE: DNA
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Artificial sequence
(ix) FEATURE:
(D) OTHER INFORMATION: oligonucleotide primer 7RC
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:67:
GCCCTTGGAG TCCAGCAGGA AGTTGGTGTA GCGGGTACAG GAGTAGCGCC AGGACATGCA 60
(2) INFORMATION FOR SEQ ID N0:68:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 60 base pairs
(B) TYPE: Nucleic acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(ii) MOLECULE TYPE: DNA
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Artificial sequence
(ix) FEATURE:
(D) OTHER INFORMATION: oligonucleotide primer 8RC
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:68:
GTCCACCTCC ACCTTGCCGC CCTTCTCGAT GATCACGGGG GAGCGCCAGC GGTACAGCTT 60
(2) INFORMATION FOR SEQ ID N0:69:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 60 base pairs
(B) TYPE: Nucleic acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(ii) MOLECULE TYPE: DNA
67
CA 02418780 2003-02-28
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Artificial sequence
(ix) FEATURE:
(D) OTHER INFORMATTON: oligonucleotide primer 9RC
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:69:
GGTCACGGGG GTGGCGGCGG AGCCGTCCAG CACCACGCGC TTCAGGTCGA TCAGGTGGCC 60
(2) INFORMATION FOR SEQ ID N0:70:
(i) SEQUENCE CHARACTERISTICS:
{A) LENGTH: 41 base pairs
(B) TYPE: Nucleic acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(ii) MOLECULE TYPE: DNA
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Artificial sequence
(ix) FEATURE:
(D) OTHER INFORMATION: oligonucleotide primer lORC
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:70:
GGCGGATCCT AGGGGCGACA CCACTGCTCG GCGGACACCT T 41
(2) INFORMATION FOR SEQ ID N0:71:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 603 base pairs
(B) TYPE: Nucleic acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(ii) MOLECULE TYPE: DNA
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Artificial sequence
(ix) FEATURE:
(A) NAME/KEY:CDS
(B) LOCATION: (1)...(603)
(D) OTHER INFORMATION: synORFS variant2 of IAF-Klop strain of PRRSV
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:71:
ATG CTG GGC AAG TGC CTG ACC GCC GGC TGT TGC TCC CAG CTG CCC TTC 48
Met Leu Gly Lys Cys Leu Thr Ala Gly Cys Cys Ser Gln Leu Pro Phe
1 5 10 15
CTG TGG TGT ATC GTG CCC TTC TGT TTC GCC GCC CTG GTG AAC GCC TCC 96
Leu Trp Cys Ile Val Pro Phe Cys Phe Ala Ala Leu Val Asn Ala Ser
20 25 30
68
CA 02418780 2003-02-28
TCGTCCTCG TCGTCCCAG CTGCAGTCC ATCTAC CTG ACCATCTGT 144
AAC
SerSerSer SerSerGln LeuGlnSer IleTyrAsnLeu ThrIleCys
35 40 45
GAGCTGAAC GGCACCGAC TGGCTGAAC AAGAACTTCGAC TGGGCCGTG 192
GluLeuAsn GlyThrAsp TrpLeuAsn LysAsnPheAsp TrpAlaVal
50 55 60
GAGACCTTC GTGATCTTC CCCGTGCTG ACCCACATCGTG TCCTACGGC 240
GluThrPhe ValIlePhe ProValLeu ThrHisIleVal SerTyrGly
65 70 75 80
GCCCTGACC ACCTCCCAC TTCCTGGAC GCCGTGGGCCTG ATCACCGTG 288
AlaLeuThr ThrSerHis PheLeuAsp AlaValGlyLeu IleThrVal
85 90 95
TCCACCGCC GGCTACTAC CACGGCCGC TACGTACTGTCC TCCGTGTAC 336
SerThrAla GlyTyrTyr HisGlyArg TyrValLeuSer SerValTyr
100 105 110
GCCGTGTGC GCCCTGGCC GCCCTGATC TGCTTCGTGATC CGCCTGACA 384
AlaValCys AlaLeuAla AlaLeuIle CysPheValIle ArgLeuThr
115 120 125
AAGAACTGC ATGTCCTGG CGCTACTCC TGTACACGCTAC ACCAACTTC 432
LysAsnCys MetSerTrp ArgTyrSer CysThrArgTyr ThrAsnPhe
130 135 140
CTGCTGGAC TCCAAGGGC AAGCTGTAC CGCTGGCGCTCC CCCGTGATC 480
LeuLeuAsp SerLysGly LysLeuTyr ArgTrpArgSer ProValIle
14S 150 155 160
ATCGAGAAG GGCGGCAAG GTGGAGGTG GACGGCCACCTG ATCGACCTG 528
IleGluLys GlyGlyLys ValGluVal AspGlyHisLeu IleAspLeu
165 170 175
AAGCGCGTG GTGCTGGAC GGCTCCGCC GCCACCCCCGTG ACCAAGGTG 576
LysArgVal ValLeuAsp GlySerAla AlaThrProVal ThrLysVal
180 185 190
TCCGCCGAG CAGTGGTGT CGCCCCTAG 603
SerAlaGlu GlnTrpCys ArgPro
195 200
2) INFORMATION FOR SEQ ID N0:72:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH:200 amino acids
(B) TYPE: Amino Acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Protein
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Artificial Sequence
(ix) FEATURE:
(D) OTHER INFORMATION: synORF5 variant2 of IAF-Klop strain of PRRSV
69
CA 02418780 2003-02-28
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:72:
Met Leu Gly Lys Cys Leu Thr Ala Gly Cys Cys Ser Gln Leu Pro Phe
1 5 10 15
Leu Trp Cys Ile Val Pro Phe Cys Phe Ala Ala Leu Val Asn Ala Ser
20 25 30
Ser Ser Ser Ser Ser Gln Leu Gln Ser Ile Tyr Asn Leu Thr Ile Cys
35 40 45
Glu Leu Asn Gly Thr Asp Trp Leu Asn Lys Asn Phe Asp Trp Ala Val
50 55 60
Glu Thr Phe Val Ile Phe Pro Val Leu Thr His Ile Val Ser Tyr Gly
65 70 75 80
Ala Leu Thr Thr Ser His Phe Leu Asp Ala Val Gly Leu Ile Thr Val
85 90 95
Ser Thr Ala Gly Tyr Tyr His Gly Arg Tyr Val Leu Ser Ser Val Tyr
100 105 110
Ala Val Cys Ala Leu Ala Ala Leu Ile Cys Phe Val Ile Arg Leu Thr
115 120 125
Lys Asn Cys Met Ser Trp Arg Tyr Ser Cys Thr Arg Tyr Thr Asn Phe
130 135 140
Leu Leu Asp Ser Lys Gly Lys Leu Tyr Arg Trp Arg Ser Pro Val Ile
145 150 155 160
Ile Glu Lys Gly Gly Lys Val Glu Val Asp Gly His Leu Ile Asp Leu
165 170 175
Lys Arg Val Val Leu Asp Gly Ser Ala Ala Thr Pro Val Thr Lys Val
180 1B5 190
Ser Ala Glu Gln Trp Cys Arg Pro
195 200
