U.S. patent application number 12/488475 was filed with the patent office on 2010-01-14 for porcine adenovirus e1 and e4 regions.
Invention is credited to SURESH K. TIKOO.
Application Number | 20100008947 12/488475 |
Document ID | / |
Family ID | 26894887 |
Filed Date | 2010-01-14 |
United States Patent
Application |
20100008947 |
Kind Code |
A1 |
TIKOO; SURESH K. |
January 14, 2010 |
PORCINE ADENOVIRUS E1 AND E4 REGIONS
Abstract
The present invention relates to the characterization of the
porcine adenovirus E1 and E4 regions. The complete nucleotide
sequence of the genome of porcine adenovirus type 3 (PAV-3),
providing the characterization of the PAV3 E1 region, is described
herein. Methods for construction of infectious PAV genomes by
homologous recombination in procaryotic cells are provided.
Recombinant PAV viruses are obtained by transfection of mammalian
cells with recombinant PAV genomes. The PAV-3 genome can be used as
a vector for the expression of heterologous nucleotide sequences,
for example, for the preparation and administration of subunit
vaccines to swine or other mammals.
Inventors: |
TIKOO; SURESH K.;
(SASKATOON, CA) |
Correspondence
Address: |
MORRISON & FOERSTER LLP
755 PAGE MILL RD
PALO ALTO
CA
94304-1018
US
|
Family ID: |
26894887 |
Appl. No.: |
12/488475 |
Filed: |
June 19, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10199550 |
Jul 19, 2002 |
7569217 |
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12488475 |
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09963038 |
Sep 24, 2001 |
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10199550 |
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Current U.S.
Class: |
424/199.1 |
Current CPC
Class: |
A01K 2227/108 20130101;
C12N 7/00 20130101; C12N 2710/10343 20130101; C07K 14/005 20130101;
C12N 2710/10322 20130101; C12N 2710/10361 20130101; C12N 15/86
20130101 |
Class at
Publication: |
424/199.1 |
International
Class: |
A61K 39/00 20060101
A61K039/00 |
Claims
1. A composition capable of inducing an immune response in an
animal subject, said composition comprising a replication-defective
recombinant PAV vector, wherein the PAV vector lacks E1A function
and retains E1B.sup.small function and wherein the vector comprises
a heterologous nucleotide sequence that encodes an immunogenic
polypeptide; and a pharmaceutically acceptable vehicle.
2. The composition of claim 1, wherein the animal subject is a
mammalian subject.
3. A composition capable of inducing an immune response in an
animal subject, said composition comprising a replication-defective
recombinant PAV vector, wherein the vector lacks E1B.sup.large
function and wherein the vector comprises a heterologous nucleotide
sequence that encodes an immunogenic polypeptide; and a
pharmaceutically acceptable vehicle.
4. The composition of claim 3, wherein the animal subject is a
mammalian subject.
5. A composition capable of inducing an immune response in an
animal subject, said composition comprising a replication-defective
recombinant PAV vector, wherein the PAV vector lacks E4 ORF3
function and wherein the vector comprises a heterologous nucleotide
sequence that encodes an immunogenic polypeptide; and a
pharmaceutically acceptable vehicle.
6. The composition of claim 5, wherein the animal subject is a
mammalian subject.
7. The composition of claim 2, wherein said immunogenic polypeptide
is a pathogen antigen.
8. The composition of claim 4, wherein said immunogenic polypeptide
is a pathogen antigen.
9. The composition of claim 6, wherein said immunogenic polypeptide
is a pathogen antigen.
10. A method for eliciting an immune response in a mammalian host
comprising administering a composition according to claim 2 to said
host.
11. A method for eliciting an immune response in a mammalian host
comprising administering a composition according to claim 4 to said
host.
12. A method for eliciting an immune response in a mammalian host
comprising administering a composition according to claim 6 to said
host.
13. A vaccine for protecting an animal host against infection
comprising a replication-defective PAV, wherein the PAV vector
lacks E1A function and retains E1B.sup.small function and wherein
said heterologous nucleotide sequence encodes an immunogenic
polypeptide.
14. The vaccine of claim 13, wherein the animal host is a mammalian
host.
15. A vaccine for protecting an animal host against infection
comprising a replication-defective PAV, wherein the vector lacks
E1B.sup.small function and wherein said heterologous nucleotide
sequence encodes an immunogenic polypeptide.
16. The vaccine of claim 15, wherein the animal host is a mammalian
host.
17. A vaccine for protecting an animal host against infection
comprising a replication-defective PAV, wherein the PAV vector
lacks E4 ORF3 function and wherein said heterologous nucleotide
sequence encodes an immunogenic polypeptide.
18. The vaccine of claim 17, wherein the animal host is a mammalian
host.
19. A composition capable of inducing an immune response in an
animal subject, said composition comprising a replication-defective
recombinant PAV vector, wherein the PAV vector lacks E1A function
and E1B.sup.large function and wherein the vector comprises a
heterologous nucleotide sequence that encodes an immunogenic
polypeptide; and a pharmaceutically acceptable vehicle.
20. The composition of claim 19, wherein the animal subject is a
mammalian subject.
21. The composition of claim 20, wherein said immunogenic
polypeptide is a pathogen antigen.
22. A method for eliciting an immune response in a mammalian host
comprising administering a composition according to claim 20 to
said host.
23. The composition of claim 19, wherein the PAV vector further
lacks E1B.sup.small function.
24. The composition of claim 20, wherein the PAV vector further
lacks E1B.sup.small function.
25. The composition of claim 24, wherein said immunogenic
polypeptide is a pathogen antigen.
26. A method for eliciting an immune response in a mammalian host
comprising administering a composition according to claim 24 to
said host.
27. A vaccine for protecting an animal host against infection
comprising a replication-defective PAV, wherein the PAV vector
lacks E1A function and E1B.sup.large function and wherein said
heterologous nucleotide sequence encodes an immunogenic
polypeptide.
28. The vaccine of claim 27, wherein the animal host is a mammalian
host.
29. The vaccine of claim 27, wherein the PAV vector further lacks
E1B.sup.small function.
30. The vaccine of claim 28, wherein the PAV vector further lacks
E1B.sup.small function.
Description
CROSS-REFERENCED TO RELATED APPLICATIONS
[0001] This is a divisional application of U.S. patent application
Ser. No. 10/199,550, filed Jul. 19, 2002, which is a
continuation-in-part application of U.S. patent application Ser.
No. 09/963,038, filed Sep. 24, 2001, which are incorporated by
reference herein in their entirety.
TECHNICAL FIELD
[0002] The present invention is in the field of recombinant
mammalian viral vectors. More particularly, it concerns recombinant
porcine adenovirus vectors for diagnostic and therapeutic purposes,
such as for vaccines, gene delivery and expression systems.
BACKGROUND
[0003] Adenoviruses are double-stranded DNA viruses that have been
isolated from a wide variety of avian and mammalian species,
including swine. Porcine adenoviruses (PAV) belong to the
Mastadenovirus genus of Adenoviridae family. Of the five serotypes
identified till date (Derbyshire et al., 1975, J. Comp. Pathol.
85:437-443; Hirahara et al., 1990, Japanese J. Vet Sci.
52:407-409), serotype 3 (PAV-3) could propagate to high titers in
cell culture. While the majority of adenovirus infections in swine
are subclinical, porcine adenovirus (PAV) infection has been
associated with encephalitis, pneumonia, kidney lesions and
diarrhea. Derbyshire (1992) In: "Diseases of Swine" (ed. Leman et
al.), 7th edition, Iowa State University Press, Ames, Iowa. pp.
225-227. Thus, there is a need for vaccines that will provide
protection against PAV infection.
[0004] In addition to their potential ability to provide protection
against PAV infection, PAVs could also be used as viral vaccine
vectors, if insertion capacity can be determined, and appropriate
insertion sites can be defined and characterized. It has been shown
that PAV is capable of stimulating both humoral response and a
mucosal antibody responses in the intestine of infected piglets.
Tuboly et al. (1993) Res. in Vet. Sci. 54:345-350. Thus,
recombinant PAV vaccine vectors would be especially useful, as they
would be likely to be capable of providing both systemic and
mucosal immunity to antigens encoded by native and/or recombinant
PAV genomes.
[0005] Cross-neutralization studies have indicated the existence of
at least five serotypes of PAV. Derbyshire et al. (1975) J. Comp.
Pathol. 85:437-443; and Hirahara et al. (1990) Jpn. J. Vet. Sci.
52:407-409. Previous studies of the PAV genome have included the
determination of restriction maps for PAV Type 3 (PAV-3) and
cloning of restriction fragments representing the complete genome
of PAV-3. Reddy et al. (1993) Intervirology 36:161-168. In
addition, restriction maps for PAV-1 and PAV-2 have been
determined. Reddy et al. (1995b) Arch. Virol. 140:195-200.
[0006] Nucleotide sequences have been determined for segments of
the genome of various PAV serotypes. The transcription map and
complete DNA sequence of PAV-3 genome was reported (Reddy et al.,
1998, Virus Res. 58:97-106 and Reddy et al., 1998, Virology
251:414-426). Sequences of the E3, pVIII and fiber genes of PAV-3
were determined by Reddy et al. (1995a) Virus Res. 36:97-106. The
E3, pVIII and fiber genes of PAV-1 and PAV-2 were sequenced by
Reddy et al. (1996) Virus Res. 43:99-109; while the PAV-4 E3, pVIII
and fiber gene sequences were determined by Kleiboeker (1994) Virus
Res. 31:17-25. The PAV-4 fiber gene sequence was determined by
Kleiboeker (1995b) Virus Res. 39:299-309. Inverted terminal repeat
(ITR) sequences for all five PAV serotypes (PAV-1 through PAV-5)
were determined by Reddy et al. (1995c) Virology 212:237-239. The
PAV-3 penton sequence was determined by McCoy et al. (1996a) Arch.
Virol. 141:1367-1375. The nucleotide sequence of the E1 region of
PAV-4 was determined by Kleiboeker (1995a) Virus Res. 36:259-268.
The sequence of the protease (23K) gene of PAV-3 was determined by
McCoy et al. (1996b) DNA Seq. 6:251-254. The sequence of the PAV-3
hexon gene (and the 14 N-terminal codons of the 23K protease gene)
has been deposited in the GenBank database under accession No.
U34592. The unpublished sequence of the PAV-3 100K gene has been
deposited in the GenBank database under accession No. U82628. The
sequence of the PAV-3 E4 region has been determined by Reddy et al.
(1997) Virus Genes 15:87-90.
[0007] Adenoviruses have proven to be effective vectors for the
delivery and expression of foreign genes in a number of specific
applications, and have a number of advantages as potential gene
transfer and vaccine vectors. See Gerard et al (1993) Trends
Cardiovasc. Med. 3:171-177; Imler et al. (1995) Hum. Gene Ther.
6:711-721. The ability of these vectors to mediate the efficient
expression of candidate therapeutic or vaccine genes in a variety
of cell types, including post mitotic cells, is considered an
advantage over other gene transfer vectors. Adenoviral vectors are
divided into helper-independent and helper-dependent groups based
on the region of the adenoviral genome used for the insertion of
transgenes. Helper-dependent vectors are usually made by deletion
of E1 sequences and substitution of foreign DNA, and are produced
in complementing human cell lines that constitutively express E1
proteins. Graham et al. (1977) J. Gen. Virol. 36:59-74; Fallaux et
al. (1996) Hum. Gene Ther. 7:215-222; Fallaux et al. (1998) Hum.
Gene Ther. 9:1909-1917. However, porcine adenoviruses do not
replicate in human cell lines; hence these lines are unsuitable for
the propagation of E1-deleted PAV vectors. E1A region is described
in Darbyshire (1966, Nature 211:102) and Whyte et al., 1988, J.
Virol. 62:257-265.
[0008] Though E1-deleted viruses do not replicate in cells that do
not express E1 proteins, the viruses can express foreign proteins
in these cells, provided the genes are placed under the control of
a constitutive promoter. Xiang et al. (1996) Virology 219:220-227.
Vaccination of animals with adenovirus recombinants containing
inserts in the E1 region induced a systemic immune response and
provided protection against subsequent challenge. Imler et al
(1995) Hum. Gene Ther. 6:711-721; Imler et al. (1996) Gene Therap
3:75-84. This type of expression vector provides a significant
safety profile to the vaccine as it eliminates the potential for
dissemination of the vector within the vaccine and therefore, the
spread of the vector to non-vaccinated contacts or to the general
environment. However, the currently used human adenovirus (HAV)
based vectors are endemic in most populations, which provides an
opportunity for recombination between the helper-dependent viral
vectors and wild type viruses. To circumvent some of the problems
associated with the use of human adenoviruses, non human
adenoviruses have been explored as possible expression vectors.
[0009] Use of vectors containing an intact E1 region for gene
therapy in humans and vaccination in animals is unsafe because they
have the ability to replicate in normal cells and spread to other
animals, and they retain any oncogenic potential of the E1 region.
WO 99/53047 disclose the use of PAV vectors deleted in their E1
region. See Klonjkowski et al (1997) Hum. Gene Ther. 8:2103-2115
which discloses E1 deleted canine adenovirus 2.
[0010] There remains a need for improved adenoviral vectors for
expression of transgenes in mammalian cells, and for the
development of effective recombinant PAV vectors for use in
immunization and expression systems.
SUMMARY OF THE INVENTION
[0011] The present invention relates to the characterization of the
porcine adenovirus E1 and E4 regions. The present invention
discloses the complete nucleotide sequence of the genome of porcine
adenovirus type 3 (PAV-3) and provides the characterization of the
PAV3 E1 region, including E1A, EB.sup.small, E1B.sup.large and E4
region ORF1-ORF7. As shown herein, E1A, E1B.sup.large and E4 ORF3
are essential for replication of PAV3. Nucleic acid sequences that
are substantially homologous to those comprising a PAV genome are
also encompassed by the invention. Substantially homologous
sequences include those capable of duplex and/or triplex formation
with a nucleic acid comprising all or part of a PAV genome (or with
its complement). As is known to those of skill in the art, duplex
formation is influenced by hybridization conditions, particularly
hybridization stringency. Factors affecting hybridization
stringency are well-known to those of skill in the art. See, for
example, Sambrook et al. (1989) Molecular Cloning: A Laboratory
Manual; Hames et al. 1985) Nucleic Acid Hybridisation: A Practical
Approach, IRL Press Ltd., Oxford Accordingly, it is within the
skill of the art to identify a sequence that is substantially
homologous to a sequence from a PAV genome.
[0012] In particular, the present invention provides a
replication-defective recombinant PAV vector, comprising at least
one heterologous nucleotide sequence, wherein the PAV vector lacks
E1A and/or E1B.sup.large function and retains E1B.sup.small
function. In some embodiments, the vector comprises a deletion of
part or all of the E1A and/or E1B.sup.large gene region. In other
embodiments, the vector comprises an insertion in the E1A and/or
E1B.sup.large gene region that inactivates the E1A and/or
E1B.sup.large region function. In some embodiments, the vector
further comprises a deletion of part or all of the E3 region, or
other essential or non-essential regions of the adenovirus. In
additional embodiments, the PAV is PAV3.
[0013] In yet other embodiments, the present invention provides a
replication-defective recombinant PAV vector that comprises a
deletion in the E1 region that consists of a deletion of the E1A
and/or E1B.sup.large region. In yet other embodiments, the present
invention provides a replication-defective recombinant PAV vector
that comprises an insertion in the E1 region that consists of an
insertion in the E1A and/or E1B.sup.large region that inactivates
E1A and/or E1B.sup.large region function.
[0014] The present invention also provides a replication-defective
recombinant PAV vector comprising at least one heterologous
nucleotide sequence, wherein the PAV vector lacks E1A function and
E1B.sup.small function and retains E1B.sup.large function. In some
embodiments, the vector comprises a deletion of part or all of the
E1A and E1B.sup.small regions. In other embodiments, the vector
comprises an insertion that inactivates the E1A or E1B.sup.small
gene region function. In further embodiments, the vector has a
deletion of part or all of the E3 region, and/or part or all of
non-essential E4 region and/or or other non-essential regions of
the adenovirus.
[0015] In further embodiments, the present invention provides a PAV
vector comprising at least one heterologous nucleotide sequence,
wherein said vector lacks E1B.sup.small function and retains E1A
and E1B.sup.large function. In some embodiments, the vector
comprises a deletion of part or all of the E1B.sup.small region. In
further embodiments, the vector comprises a deletion in the E3
region or other non-essential regions. In additional embodiments,
the PAV is PAV3.
[0016] In other embodiments, the present invention provides a
replication-defective PAV vector that lacks E4 ORF3 function. In
some examples, the vector comprises a deletion of part or all of
the E4 ORF3 region. In some examples, the vector comprises an
insertion in the E4 ORF3 region that inactivates E4 ORF3.
[0017] In further embodiments, the heterologous nucleotide sequence
encodes a therapeutic polypeptide. In yet further embodiments, the
heterologous polypeptide sequence encodes an antigen. In yet
further embodiments, the therapeutic polypeptide is selected from
the group consisting of coagulation factors, growth hormones,
cytokines, lymphokines, tumor-suppressing polypeptides, cell
receptors, ligands for cell receptors, protease inhibitors,
antibodies, toxins, immunotoxins, dystrophins, cystic fibrosis
transmembrane conductance regulator (CFTR), immunogenic
polypeptides and vaccine antigens.
[0018] The present invention also provides host cells infected with
a recombinant PAV vector of the present invention. The present
invention also provides methods for producing a recombinant PAVs
that comprises introducing a PAV vector that lacks E1A function
and/or E1B.sup.large function and retains E1B.sup.small function
into a helper cell line that expresses E1A function and/or
E1B.sup.large function and recovering virus from the infected
cells. In one embodiment, the present invention comprises
introducing a PAV vector that lacks E1A function, and retains
E1B.sup.small and E1B.sup.large function, into a helper cell line
that expresses E1A function. In some embodiments, the helper cell
line expresses human E1A function.
[0019] The present invention also provides recombinant mammalian
cell lines that comprise nucleic acid encoding mammalian adenovirus
E1A function and lack nucleic acid encoding mammalian adenovirus
E1B.sup.small function. In some embodiments, the E1A function is
human E1A function. The present invention also provides recombinant
mammalian cell lines that comprise nucleic acid encoding mammalian
adenovirus E1B.sup.large function and lack nucleic acid encoding
mammalian adenovirus E1B.sup.small function. In some embodiments,
the E1B.sup.large function is human E1B.sup.large function. In
other embodiments, the helper cell line expresses porcine
E1B.sup.large function. In some embodiments, the cell line is of
porcine origin. The present invention also provides methods for
producing a recombinant PAV that lacks E1A and retains
E1B.sup.small function. The present invention also provides
recombinant mammalian cell lines that comprise nucleic acid
encoding porcine E4 ORF3 function.
[0020] In some embodiments, the present invention provides a method
comprising introducing, into an appropriate helper cell line, a
porcine adenovirus vector comprising ITR sequences, PAV packaging
sequences, and at least one heterologous nucleotide sequence,
wherein said vector lacks E1A and/or E1B.sup.large function and
retains E1B.sup.small function; culturing the cell line under
conditions whereby adenovirus virus replication and packaging
occurs; and recovering the adenovirus from the infected cells. In
some embodiments, the PAV is PAV3. The present invention also
provides methods for producing a recombinant PAV that lacks
E1B.sup.small function and retains E1A and/or E1B.sup.large
function.
[0021] The present invention provides viral particles comprising a
PAV vector of the present invention. The present invention also
provides host cells comprising a PAV vector of the present
invention. In additional embodiments, the invention provides
compositions that are able to elicit an immune response or able to
provide immunity to PAV infection, through expression of antigenic
PAV polypeptides. The invention also provides vectors comprising
PAV genome sequences, including sequences encoding various PAV
genes as well as PAV regulatory sequences, which are useful for
controlling the expression of heterologous genes inserted into PAV
vectors.
BRIEF DESCRIPTION OF THE DRAWINGS
[0022] FIGS. 1-1 through 1-10 show the complete nucleotide sequence
of the PAV-3 genome (SEQ ID NO: 1).
[0023] FIG. 2 shows the transcriptional map of the PAV-3 genome
derived from alignment of the sequences of cDNA clones with the
genomic sequence, and nuclease protection mapping of viral
transcripts. The PAV-3 genome is represented by the thick
horizontal line, with the numbers below the line representing PAV-3
map units (i.e., percentage of genome length from the left end).
Rightward-reading transcription units are depicted above the line
and leftward-reading transcription units are shown below the
line.
[0024] FIGS. 3A-3B show immunoprecipitation of E1A and E1B proteins
from various cell lines. In FIG. 3A, proteins in cell lysates were
separated by gel electrophoresis, and analyzed by immunoblotting
using the DP11 monoclonal antibody, which recognizes the human
adenovirus E1A protein. Lane 1: 293 cells (human cells transformed
by HAV-5, which express adenovirus E1A and E1B); Lane 2: Fetal
porcine retinal cells; Lane 3: VIDO R1 cells; Lane 4: 293 cells. In
FIG. 3B, proteins in cell lysates were separated by gel
electrophoresis, and analyzed by immunoblotting using the DP17
monoclonal antibody, which recognizes the human adenovirus E1B
protein. Lane 1: human 293 cells; Lane 2: Fetal porcine retinal
cells; Lane 3: VIDO R1 cells; Lane 4: 293 cells.
[0025] FIG. 4 shows a map of the plasmid pPAV-101.
[0026] FIG. 5 shows a map of the plasmid pPAV-102.
[0027] FIG. 6 shows a map of the plasmid pPAV-300.
[0028] FIG. 7 shows proteins labeled after infection of VIDO R1
cells with a recombinant PAV containing the PRV gp50 gene inserted
in the E3 region. Labeled proteins were separated by gel
electrophoresis; an autoradiogram of the gel is shown. Lane 1:
Molecular weight markers of 30K, 46K, 69K and 96K, in order of
increasing molecular weight. Lane 2: Mock-infected cells, 12 hours
post-infection. Lane 3: PAV-3-infected cells, 12 hours
post-infection. Lane 4: cells infected with a recombinant PAV
containing the PRV gp50 gene, 12 hours post-infection. Lane 5:
cells infected with a recombinant PAV containing the PRV gp50 gene,
16 hours post-infection. Lane 6: cells infected with a recombinant
PAV containing the PRV gp50 gene, 24 hours post-infection.
[0029] FIG. 8 provides a schematic diagram of the construction of
an E1- and E3-deleted PAV vector with a green fluorescent protein
gene insertion.
[0030] FIGS. 9A-9F provide a schematic representation of strategies
used for generation of porcine genomic DNA in plasmids. (Figure A)
plasmid pPAVXhoIRL; (Figure B) plasmid pFPAV211; (Figure C) plasmid
pFPAV212; (Figure D) plasmid pFPAV507; (Figure E) plasmid pFPAV214;
(Figure F) plasmid pFPAV216. ITR (filled box); The origin of DNA
sequences is as follows: BAV-3 genome (open box); AmpR gene
(arrow); plasmid DNA (broken line). The plasmid maps are not drawn
to scale.
[0031] FIG. 10 shows the immunoprecipitation of proteins
synthesized by in vitro transcription and translation of plasmids.
[.sup.35S]-methionine labeled in vitro transcribed and translated
pSP64-PE1A (lanes 7,9), pSP64-PE1Bs (lanes 4,6), pSP64-PE1B1 (lanes
1,3) and pSP64polyA (lanes 2,5,8) products before (lanes 3,6,9) and
after immunoprecipitation with anti-E1A (lanes 8,9),
anti-E1B.sup.small (lanes 5,6) and anti-E1B.sup.large (lanes 2,3)
were separated on 10% SDS-PAGE gels under reducing conditions. The
positions of the molecular weight markers are shown to the left of
the panel.
[0032] FIG. 11 shows the in vivo immunoprecipitation of E1
proteins. Proteins from the lysates of [.sup.35S]
methionine-cysteine labeled mock (lane 3) or PAV3 infected (lane 1,
6 h post infection; lane 2, 24 h post infection) VIDO R1 cells were
immunoprecipitated with anti-E1A serum (panel A),
anti-E1B.sup.small serum (panel B), anti-E1B.sup.small serum (panel
C) and separated on 10% SDS-PAGE under reducing conditions. The
positions of the molecular weight markers are indicated to the left
of each panel.
[0033] FIGS. 12A-12C provide the restriction enzyme analysis of
recombinant PAV-3 genome. (Figure A) The viral DNAs were extracted
from VIDO R1 cells infected with PAV211 (lane 1), PAV212 (lane 2)
or wild-type PAV-3 (lane 3) and digested with SpeI. Sizes of marker
(M) are shown in basepairs. (Figure B) The viral DNAs were
extracted from VIDO R1 cells infected with PAV214 (lane 1) or
wild-type PAV-3 (lane 2) and digested with NheI. Sizes of marker
(M) are shown in base pairs. (Figure C) The viral DNAs were
extracted from VIDO R1 cells infected with PAV216 (lane 2) or
wild-type PAV-3 (lane 1) and digested with AseI. Sizes of marker
(M) are shown in base pairs.
[0034] FIG. 13 shows Western blot analysis of PAV-3 protein
expression in mutant infected cells. Proteins from wild-type PAV3
(lane 3), PAV211 (lane 2), or PAV212 (lane 1) infected ST cells
were separated by 12.5% SDS-PAGE under reducing conditions and
transferred to nitrocellulose. The separated proteins were probed
in Western blots by anti-E1A (panel C), anti-E1B.sup.small (panel
A) or anti-DBP (panel B). The positions of the molecular weight
markers are shown to the left of each panel.
[0035] FIG. 14 shows Western Blot analysis of GFP expression.
Proteins from purified GFP (lane 2) or mock (lane 1), wild-type
PAV-3 (lane 3) and PAV216 (lane 4 and 5) infected VIDO R1 cells
harvested at 24 h.p.i (lane 3, 4) and 48 h.p.i. (lane 5) were
separated by 10% SDS-PAGE under reducing conditions and transferred
to nitrocellulose. The separated proteins were probed Western blots
by anti-GFP polyclonal antibody.
[0036] FIGS. 15A-15B shows Virus titers of recombinant and
wild-type PAV-3. Near-confluent monolayers of VIDO R1 (Figure A) or
Swine Testicular (ST) (Figure B) cells were infected with
recombinant or wild-type PAV-3. At different time points post
infection, the cell pellets were freeze-thawed and virus was
titrated on VIDO R1 cells as described in the text.
[0037] FIGS. 16A-16B. FIG. 16A shows a map of the plasmid used for
stable transfection of the VIDO-R1 cell line. The plasmid contains
the human CMV promoter, the internal ribosomal entry site (IRES),
hygromycin B phosphotransferase gene and the gene for PAdV-3
E1B-large protein. FIG. 16B shows the total genomic DNA extracted
from hygromycin-resistant cell clones was digested with HindIII and
hybridized with the labelled 1.9 kb--HindIII fragment of
pIREShyE1BL DNA containing the E1B-large gene.
[0038] FIG. 17 shows Product of RT-PCR using DNase-treated RNA
isolated from hygromycin-resistant cell clones (lane 3 to 9) and
using PAdV-3 E1B-large specific primers. RT-PCR was run with (+) or
without (-) reverse transcriptase. C-- is a PCR on pIREShyE1BL DNA
template.
[0039] FIGS. 18A-18B show immunofluorescence of VR1BL cells.
Immunofluorescence analysis was carried out using rabbit polyclonal
antisera against PAdV-3 E1B-large protein. The parent VIDO-R1 cell
line is negative FIG. 18A. New VR1BL cell line is positive for
PAdV-3 E1B-large protein expression FIG. 18B.
[0040] FIGS. 19A-19B. FIG. 19 A shows a schematic representation of
viral DNA. The origin of DNA sequences is as follows: PAdV-3 genome
(open box); ITR (filled box); thin lines show the deletions in the
E3 and E1 regions; GFP-expressing cassette, containing human CMV
promoter, GFP gene, BGH polyA signal (hatched box). Arrow indicates
the direction of the transcription of the GFP gene. FIG. 19 B shows
a restriction enzyme analysis of viral DNA. Recombinant viruses
were rescued after transfection VR1BL cells with the full-length
viral genomic DNA, cloned in plasmids. The viral DNAs were
extracted from VR1BL cells infected with PAdV-3 (lane 1), PAV227
(lane 2), PAV219 (lane 3) digested with SpeI. Lane M is 1 kb+
marker.
[0041] FIGS. 20 A-20C. FIG. 20A shows GFP expression in PAV219
infected ST cells. To detect GFP expression by PAV219, ST (swine
testis) cells were infected with m.o.i. 1 TCID50/cell FIGS. 20B and
100 TCID50/cell FIG. 20C. 24 h.p.i. the cells were harvested and
analyzed by FACS. FIG. 20A show mock-infected ST cells.
[0042] FIG. 21 shows transduction of human cell lines. Human cell
lines were infected with PAV219 at m.o.i. 100 TCID50/cell. 24
h.p.i. the cells were harvested and GFP expression was analyzed by
FACS. Tested human cell lines: A549 lung carcinoma; 293 embryo
kidney; HeLa cervix carcinoma; Hep2 larynx carcinoma; SK-N-MC
neuroblastoma; U118-MG glioblastoma; MRC-5 lung fibroblasts; SAOS-2
osteosarcoma; K562 myelogenous leukemia; Raji Burkitt's lymphoma.
ST is a fetal porcine testis cell line.
[0043] FIGS. 22A-22C show full-length plasmids with E4 deletions.
FIG. 22A is the genomic map unit of PAV3. FIG. 22B shows the
locations of the E4 TATA box, Poly A region and the seven putative
open reading frames (ORFs). FIG. 22C shows the full-length clones
with deletions of different ORFs.
[0044] FIG. 23 shows the restriction enzyme analysis of the mutant
viruses. ST cells were infected with mutant viruses and PAV3, and
viral genomic DNAs were extracted from the infected cells. All the
viral genomic DNAs were digested with AvrII, all the expected DNA
fragment sizes generated upon digestion are shown below each of the
mutant viruses. Molecular size markers of 1 kb+ are indicated.
[0045] FIG. 24 shows the PCR analysis of mutant viruses. The
PCR-amplified products from three different sets of primers
flanking the corresponding E4 deletions are shown. The expected
sizes of amplified products generated by PCRrom PAV3 and mutant
viruses are also shown at the bottom. Molecular size markers of 1
kb+are indicated.
[0046] FIG. 25 shows the growth kinetics of PAV3 E4 mutant
viruses.
DETAILED DESCRIPTION OF THE INVENTION
[0047] The present invention provides the complete nucleotide
sequence and transcriptional map of the porcine adenovirus type 3
(PAV-3) genome and the characterization of the E1 region and E4
region of PAV3. In particular, the inventors have discovered that
E1A and E1B.sup.large regions are essential for virus replication
and E1B.sup.small is non-essential for virus replication. The
inventors have discovered that E4 ORF 3 is essential for
replication and E4 ORF1, ORF2, ORF4, ORF5, ORF6 and ORF7 are
non-essential for replication. The PAV3 nucleotide sequence
comprises a linear, double-stranded DNA molecule of about 34,094
base pairs, as shown in FIG. 1 (SEQ ID NO: 1).
Previously-determined partial sequences can be aligned with the
complete genomic sequence as shown in Table 1.
TABLE-US-00001 TABLE 1 Alignment of published PAV-3 sequences PAV
Gene(s) GenBank included Genome Accession No. within sequence
Reference coordinates L43077 ITR Reddy et al., 1995c 1-144 U24432
penton McCoy et al., 1996a 13556-15283 U34592 hexon; N-terminal
unpublished 19036-21896 14 codons of 23K (protease) gene U33016
protease (23K) McCoy et al., 1996b 21897-22676 U82628 100K
unpublished 24056-26572 U10433 E3, pVIII, fiber Reddy et al., 1995a
27089-31148 L43363 E4 Reddy et al., 1997 31064-34094
[0048] Knowledge of the PAV genome sequence is useful for both
therapeutic and diagnostic procedures. Regions suitable for
insertion and regulated expression of heterologous sequences have
been identified. These regions include, but are not limited to the
E1 region including E1A, E1B.sup.small and E1B.sup.large, E3 and E4
regions, including E4 ORF 1-ORF7 regions, and the region between
the E4 region and the right end of the genome. A heterologous
nucleotide sequence, with respect to the PAV vectors of the
invention, is one which is not normally associated with PAV
sequences as part of the PAV genome. Heterologous nucleotide
sequences include synthetic sequences. Regions encoding immunogenic
PAV polypeptides, for use in immunodiagnostic procedures, have also
been identified and are disclosed herein. These include the regions
encoding the following PAV proteins: E1A, E1B.sup.small and
E1B.sup.large, E4, including ORF1-ORF7 regions, pIX, DBP, pTP, pol,
IVa2, 52K, IIIA, pIII, pVII, pV, pX, pVI, 33K, pVIII, hexon and
fiber (see Table 2). Regions essential for viral replication, such
as E1 regions E1A and E1B.sup.small, E2A, and E4 ORF3 can be
deleted to provide attenuated strains for use as vaccines.
Nonessential regions, such as E1B.sup.small and parts of the E3 and
E4 regions, such as for example E4 ORF1-ORF2 and E4 ORF 4-ORF7 can
be deleted to provide insertion sites, or to provide additional
capacity for insertion at a site other than the deleted region.
Deletions of viral sequences can be obtained by any method known in
the art, including but not limited to restriction enzyme digestion
and ligation, oligonucleotide-mediated deletion mutagenesis, and
the like.
[0049] The practice of the present invention employs, unless
otherwise indicated, conventional microbiology, immunology,
virology, molecular biology, and recombinant DNA techniques which
are within the skill of the art. These techniques are fully
explained in the literature. See, e.g., Maniatis et al., Molecular
Cloning: A Laboratory Manual (1982); DNA Cloning: A Practical
Approach, vols. I & II (D. Glover, ed.); Oligonucleotide
Synthesis (N. Gait, ed. (1984)); Nucleic Acid Hybridization (B.
Hames & S. Higgins, eds. (1985)); Transcription and Translation
(B. Hames & S. Higgins, eds. (1984)); Animal Cell Culture (R.
Freshney, ed. (1986)); Perbal, A Practical Guide to Molecular
Cloning (1984); Ausubel, et al., Current Protocols In Molecular
Biology, John Wiley & Sons (1987, 1988, 1989, 1990, 1991, 1992,
1993, 1994, 1995, 1996); and Sambrook et al., Molecular Cloning: A
Laboratory Manual (2.sup.nd Edition); vols. I, II & III
(1989).
[0050] For general information related to mammalian adenovirus see
"Fundamental Virology", second edition, 1991, ed. B. N. Fields,
Raven Press, New York, pages 771-813; and "Fields Virology", third
edition, 1995, ed. B. N. Fields, vol. 2, pages 2111-2172.
[0051] Nucleotide Sequence, Genome Organization, and Transcription
Map of Porcine Adenovirus Type 3 (PAV-3).
[0052] The complete nucleotide sequence of PAV-3 genome is 34,094
base pairs (bp) in length and has a base composition of 31.3% G,
32.5% C, 18.3% A, and 17.9% T. Thus, the sequence of the PAV-3
genome has a G+C content of 63.8%, which is unusually high when
compared with the G+C content of many other animal adenoviruses.
The genome termini share inverted terminal repeats (ITR) of 144 bp.
Reddy et al., 1995c, supra. The organization of the genome as
determined by analysis of open reading frames (ORFs), nuclease
protection mapping, and sequencing of cDNA clones, is summarized in
Table 2 and FIG. 2. The present invention relates to the
characterization of the PAV E1 region. For PAV3, the E1A region is
from nucleotide 533 to nucleotide 1222 of FIG. 1, the E1B.sup.small
region is from nucleotide 1461 to nucleotide 2069 of FIG. 1 and the
E1B.sup.large region is from nucleotide 1829 to nucleotide 3253 of
FIG. 1. E1B.sup.small and E1B.sup.large nucleotide regions are
overlapping and are differentially transcribed. Depending upon the
intended use of the PAV vector, PAV constructs can be made
comprising a deletion of part or all of the E1B.sup.small region.
For example, if the entire E1B function is intended to be deleted,
the entire E1B nucleotide region from nucleotides 1461 to 3253 can
be deleted; or the region from nucleotides 1461 to 2069 can be
deleted (which disrupts both E1B small and E1B.sup.large function);
or the region from 1461 to 2069 and additionally, any portion of
nucleotides 2069 through 3253 can be deleted. If it is intended to
delete E1B.sup.small nucleotides while retaining E1B.sup.large
function, nucleotides 1461 to 1829 are deleted, leaving the
nucleotide region for E1B.sup.large intact.
[0053] The present invention also relates to the characterization
of the E4 regions. As shown herein in the examples, E4 ORF3 is
essential for replication. Table 5 in the examples provides
nucleotide ranges for the E4 ORF regions.
[0054] One important feature of PAV-3 genome is the presence of a
short virion associated (VA) RNA gene between the splice acceptor
sites of the precursor terminal protein (pTP) and 52 kDa protein
genes (FIG. 2). Expression of VA genes increases the kinetics of
viral replication; thereby providing the potential for higher
yields of recombinant gene products using the PAV vectors of the
invention. The locations of the signature sequences present
upstream and downstream of VA RNA genes indicate the VA RNA gene of
PAV-3 is about 126 nucleotides (nt) in length. This is somewhat
shorter than most VA RNAs, whose lengths are 163.+-.14 nts, however
shorter VA RNAs have also been reported in HAV-10 and CELO virus.
Ma et al. (1996) J. Virol. 70:5083-5099; and Chiocca et al. (1996)
J. Virol. 70:2939-2949. The VA RNA genes were not found in the
genomes of BAV-3, CAV-1, and OAV. Reddy et al. (1998) J. Virol.
72:1394-1402; Morrison et al. (1997) J. Gen. Virol. 78:873-878; and
Vrati et al. (1996) Virology 220:186-199.
[0055] In PAV-3 the major late transcript initiates at 17.7 map
units (m.u.: an adenovirus map unit is 1% of genome length,
starting from the left end of the genome). There are six
3'-coterminal families of late mRNAs, denoted L1 to L6 (see FIG.
2). All mRNAs produced from the major late promoter (MLP) contain a
tripartite leader sequence (TPL). The first portion of the TPL lies
next to the MLP and is 61 nts long. The second portion lies within
the gene coding for pol and is 68 nt in length. The third portion
is 99 nts long and is located within the gene coding for pTP. Thus
the TPL of PAV-3 is 228 nt long and is derived from three exons
located at 17.7, 20.9, and 28.1 m.u.
[0056] The MLP and TPL sequences can be used for expression of a
heterologous sequence in a recombinant PAV vector or in any other
adenoviral expression system.
TABLE-US-00002 TABLE 2 Transcriptional and Translational Features
of the PAV-3 Genome Transcription Poly(A) Region Gene start site
ATG Splice donor site Splice acceptor site signal Poly(A) addition
site E1A 229R heterogeneous 533 1043 1140 1286 1307 214R 533 1286
1307 E1B 202R 1382 1461 4085 4110, 4112 474R 1382 1829 4085 4110,
4112 pIX Pix 3377 3394 4085 4110, 4112 E2A DBP 17011c 24041c
26949c, 24714c 24793c, 24051c 22560c 22536c E2B pTP 17011c 13638c
24949c, 24714c 24793c, 13772c 4075c 4053c pol 17011c 13638c 24949c,
24714c 24793.dagger.c, 13772.dagger.c 4075c 4053c IVa2 IVa2 5867c
5711c 5699c 5441c 4075c 4053c E3 27473 28765 28793 E4 33730c 31189c
31170c L1 52K 6064 10629 9684 10606 13601 13627 IIIA 6064 11719
9684 11715 13601 13627 L2 pIII 6064 13662 9684 13662 15698* 15735
pVII 6064 15170 9684 15139 15698* 15735 L3 pV 6064 15819 9684 15793
18992 19013 pX 6064 17783 9684 17776 18992 19013 pVI 6064 18076
9684 18063 18992 19013 L4 Hexon 6064 19097 9684 19096 22544 22567
Protease 6064 21934 9684 21931.dagger. 22544 22567 L5 100k 6064
24056 9684 24056 28765 28793 33K 6064 26181 9684 26130 28765 29793
pVIII 6064 27089 9684 26792 28765 28793 L6 Fiber 6064 28939 9684
28910 31143 31164 Notes: *TTGTTT is present as a polyadenylation
signal instead of AATAAA .dagger.The splice acceptor sites for the
pol and protease genes were determined based on consensus splice
acceptor sequences "c" refers to sequences on the complementary
(leftward-reading) strand of the PAV genome.
[0057] Construction of Recombinant PAV Vectors
[0058] In one embodiment of the invention, a recombinant PAV vector
is constructed by in vivo recombination between a plasmid and a PAV
genome. Generally, heterologous sequences are inserted into a
plasmid vector containing a portion of the PAV genome, which may or
may not possess one or more deletions of PAV sequences. The
heterologous sequences are inserted into the PAV insert portion of
the plasmid vector, such that the heterologous sequences are
flanked by PAV sequences that are adjacent on the PAV genome. The
PAV sequences serve as "guide sequences," to direct insertion of
the heterologous sequences to a particular site in the PAV genome;
the insertion site being defined by the genomic location of the
guide sequences.
[0059] The vector is generally a bacterial plasmid, allowing
multiple copies of the cloned sequence to be produced. In one
embodiment, the plasmid is co-transfected, into an appropriate host
cell, with a PAV genome comprising a full-length or nearly
full-length PAV genomic sequence. The PAV genome can be isolated
from PAV virions, or can comprise a PAV genome that has been
inserted into a plasmid, using standard techniques of molecular
biology and biotechnology. Construction of a plasmid containing a
PAV genome is described in Example 2, infra. Nearly full-length PAV
genomic sequences can be deleted in regions such as E1, E3, E4 and
the region between E4 and the right end of the genome, but will
retain sequences required for replication and packaging. PAV
genomes can be deleted in essential regions, such as E1A and/or
E1B.sup.large and/or E4 ORF3 if the essential function are supplied
by a helper cell line.
[0060] Insertion of the cloned heterologous sequences into a viral
genome occurs by in vivo recombination between a plasmid vector
(containing heterologous sequences flanked by PAV guide sequences)
and a PAV genome following co-transfection into a suitable host
cell. The PAV genome contains inverted terminal repeat (ITR)
sequences required for initiation of viral DNA replication (Reddy
et al. (1995c), supra), and sequences involved in packaging of
replicated viral genomes. Adenovirus packaging signals generally
lie between the left ITR and the E1A promoter. Incorporation of the
cloned heterologous sequences into the PAV genome thus places the
heterologous sequences into a DNA molecule containing viral
replication and packaging signals, allowing generation of multiple
copies of a recombinant PAV genome that can be packaged into
infectious viral particles. Alternatively, incorporation of the
cloned heterologous sequences into a PAV genome places these
sequences into a DNA molecule that can be replicated and packaged
in an appropriate helper cell line. Multiple copies of a single
sequence can be inserted to improve yield of the heterologous gene
product, or multiple heterologous sequences can be inserted so that
the recombinant virus is capable of expressing more than one
heterologous gene product. The heterologous sequences can contain
additions, deletions and/or substitutions to enhance the expression
and/or immunological effect of the expressed gene product(s).
[0061] Attachment of guide sequences to a heterologous sequence can
also be accomplished by ligation in vitro. In this case, a nucleic
acid comprising a heterologous sequence flanked by PAV guide
sequences can be co-introduced into a host cell along with a PAV
genome, and recombination can occur to generate a recombinant PAV
vector. Introduction of nucleic acids into cells can be achieved by
any method known in the art, including, but not limited to,
microinjection, transfection, electroporation, CaPO.sub.4
precipitation, DEAE-dextran, liposomes, particle bombardment,
etc.
[0062] In one embodiment of the invention, a recombinant PAV
expression cassette can be obtained by cleaving a wild-type PAV
genome with an appropriate restriction enzyme to produce a PAV
restriction fragment representing, for example, the left end or the
right end of the genome comprising E1 or E3 gene region sequences,
respectively. The PAV restriction fragment can be inserted into a
cloning vehicle, such as a plasmid, and thereafter at least one
heterologous sequence (which may or may not encode a foreign
protein) can be inserted into the E1 or E3 region with or without
an operatively-linked eukaryotic transcriptional regulatory
sequence. The recombinant expression cassette is contacted with a
PAV genome and, through homologous recombination or other
conventional genetic engineering methods, the desired recombinant
is obtained. In the case wherein the expression cassette comprises
the E1 essential regions, such as, E1A and/or E1B.sup.large or some
other essential region, such as E4 ORF3, recombination between the
expression cassette and a PAV genome can occur within an
appropriate helper cell line such as, for example, an E1A
transformed cell line when E1A region is deleted or E1A function is
inactivated, an E1B.sup.large transformed cell line when
E1B.sup.small is deleted or E1B.sup.large function is inactivated
or an E4 ORF 3 cell line when E4 ORF3 is deleted or E4 ORF3
function is inactivated. Restriction fragments of the PAV genome
other than those comprising the E1 or E3 regions are also useful in
the practice of the invention and can be inserted into a cloning
vehicle such that heterologous sequences can be inserted into the
PAV sequences. These DNA constructs can then undergo recombination
in vitro or in vivo, with a PAV genome either before or after
transformation or transfection of an appropriate host cell.
[0063] The invention also includes an expression system comprising
a porcine adenovirus expression vector wherein a heterologous
nucleotide sequence, e.g. DNA, replaces part or all of the E3
region, part or all of the E1 region, part or all of the E2 region,
part or all of the E4 region, part or all of the late region and/or
part or all of the regions occupied by the pIX, DBP, pTP, pol,
IVa2, 52K, IIIA, pIII, pVII, pV, pX, pVI, and 33K genes. The
expression system can be used wherein the foreign nucleotide
sequences, e.g. DNA, are optionally in operative linkage with a
eukaryotic transcriptional regulatory sequence. PAV expression
vectors can also comprise inverted terminal repeat (ITR) sequences
and packaging sequences.
[0064] The PAV E1A, E1B.sup.large, E4 ORF3, pIX, DBP, pTP, pol,
IVa2, 52K, IIIA, pIII, pVII, pV, pX, pVI, and 33K genes are
essential for viral replication. Therefore, PAV vectors comprising
deletions in any of these genes, or which lack functions encoded by
any of these genes, are grown in an appropriate complementing cell
line (i.e., a helper cell line). E1B.sup.small and most, if not
all, of the open reading frames in the E3 and E4 regions, e.g.
ORF1, ORF2 and ORF4-ORF7 of PAV-3 are non-essential for viral
replication and, therefore, deletions in these regions can be
constructed for insertion or to increase vector capacity, without
necessitating the use of a helper cell line for growth of the viral
vector.
[0065] In another embodiment, the invention provides a method for
constructing a full-length clone of a PAV genome by homologous
recombination in vivo. In this embodiment, two or more plasmid
clones, containing overlapping segments of the PAV genome and
together covering the entire genome, are introduced into an
appropriate bacterial host cell. Approximately 30 base pairs of
overlap is required for homologous recombination in E. coli.
Chartier et al. (1996) J. Virol. 70:4805-4810. Through in vivo
homologous recombination, the PAV genome segments are joined to
form a full-length PAV genome. In a further embodiment, a
recombinant plasmid containing left-end sequences and right-end
sequences of the PAV genome, separated by a unique restriction
site, is constructed. This plasmid is digested with the restriction
enzyme recognizing the unique restriction site, to generate a
unit-length linear plasmid, which is introduced into a cell
together with a full-length PAV genome. Homologous recombination
within the cell will result in production of a recombinant plasmid
containing a full-length PAV genome. Recombinant plasmids will also
generally contain sequences specifying replication in a host cell
and one or more selective markers, such as, for example, antibiotic
resistance.
[0066] Suitable host cells include any cell that will support
recombination between a PAV genome and a plasmid containing PAV
sequences, or between two or more plasmids, each containing PAV
sequences. Recombination is generally performed in procaryotic
cells, such as E. coli, while transfection of a plasmid containing
a viral genome, to generate virus particles, is conducted in
eukaryotic cells, preferably mammalian cells, most preferably
porcine cell cultures. The growth of bacterial cell cultures, as
well as culture and maintenance of eukaryotic cells and mammalian
cell lines are procedures which are well-known to those of skill in
the art.
[0067] In one embodiment of the invention, a replication-defective
recombinant PAV vector is used for expression of heterologous
sequences. In some embodiments, the replication-defective vector
lacks E1A and/or E1B.sup.large and/or E4 ORF3 region function. In
some embodiments, the replication-defective PAV vector comprises a
deletion of the E1A region or an inactivation of the E1A gene
function, such as through an insertion in the E1A gene region.
Construction of a deletion in the E1 region of PAV is described in
Example 3 and Example 10, infra. Heterologous sequences can be
inserted so as to replace the deleted E1A or E1B region(s), and/or
can be inserted at other sites in the PAV genome, preferably E3, E4
and/or the region between E4 and the right end of the genome.
Replication-defective vectors with deletions in essential E1
regions, such as, E1A and E1B.sup.large are grown in helper cell
lines expressing E1A and E1B.sup.large, which provide the deleted
E1 function. Replication-defective vectors with deletions in E4
ORF3 are grown in helper cell lines expressing E4 ORF3.
[0068] Accordingly, in one embodiment of the invention, a number of
recombinant helper cell lines are produced according to the present
invention by constructing an expression cassette comprising an
adenoviral essential E1 region, such as E1A and/or E1B.sup.large
and/or E4 ORF3 and transforming host cells therewith to provide
complementing cell lines or cultures providing deleted functions.
In some embodiments, the host cell is transformed with a human or
porcine E1A gene region. In other embodiments, the host cell is
transformed with human or porcine E1B gene region. In other
embodiments, the host cell is transformed with human or porcine E4
ORF3 gene region. The terms "complementing cell," "complementing
cell line," "helper cell" and "helper cell line" are used
interchangeably herein to denote a cell line that provides a viral
function that is deficient in a deleted PAV, including an essential
E1 function or essential E4 function. These recombinant
complementing cell lines are capable of allowing a
replication-defective recombinant PAV, having a deleted E1 gene
region that is essential for replication, such as E1A and E1B
wherein the deleted sequences are optionally replaced by
heterologous nucleotide sequences, to replicate and express one or
more foreign genes or fragments thereof encoded by the heterologous
nucleotide sequences. PAV vectors with E1 deletions, wherein
heterologous sequences are inserted in regions other than E1, can
also be propagated in these complementing cell lines, and will
express the heterologous sequences if they are inserted downstream
of a PAV promoter or are inserted in operative linkage with a
eukaryotic regulatory sequence. Helper cell lines include VIDO R1
cells, as described in Example 1, infra. Briefly, the VIDO R1 cell
line is a porcine fetal retinal cell line that has been transfected
with DNA from the human adenovirus type 5 (HAV-5) E1 region, and
which supports the growth of PAV E1A deletions and HAV-5 E1
deletions. Recombinant complementing cell lines expressing E4 ORF3
are capable of allowing a replication-defective recombinant PAV,
having a deleted E4 ORF3 gene region that is essential for
replication and optionally replaced by heterologous nucleotide
sequences, to replicate and express one or more foreign genes or
fragments thereof encoded by the heterologous nucleotide
sequences.
[0069] In the present invention, a PAV E1-complementing cell line
employing the E1 region of HAV-5 is shown to complement PAV-3 E1
mutants. There are several reasons that the E1 region of HAV-5 was
used for transformation of porcine embryonic retinal cells. The E1
region of HAV-5 was shown to transform human retina cells very
efficiently. Fallaux et al. (1998) supra. The E1 region of HAV-5
has been thoroughly characterized and the monoclonal antibodies
against the E1 proteins are readily available from commercial
sources. In addition, the E1A region of HAV-5 was shown to
complement the E1A functions of several non-human adenoviruses.
Ball et al. (1988) J. Virol. 62:3947-3957; Zheng et al. (1994)
Virus Res. 31:163-186. As shown herein in Example 11, a helper cell
line expressing human adenovirus E1 and porcine E1B.sup.large was
able to rescue a porcine adenovirus having a deletion of the entire
E1 region, including E1B.sup.large nucleic acid.
[0070] More generally, replication-defective recombinant PAV
vectors, lacking one or more essential functions encoded by the PAV
genome, can be propagated in appropriate complementing cell lines,
wherein a particular complementing cell line provides a function or
functions that is (are) lacking in a particular defective
recombinant PAV vector. Complementing cell lines can provide viral
functions through, for example, co-infection with a helper virus,
or by integrating or otherwise maintaining in stable form a
fragment of a viral genome encoding a particular viral
function.
[0071] In another embodiment of the invention, E1 function (or the
function of any other viral region which may be mutated or deleted
in any particular viral vector) can be supplied (to provide a
complementing cell line) by co-infection of cells with a virus
which expresses the function that the vector lacks.
[0072] PAV Expression Systems
[0073] In one embodiment, the present invention identifies and
provides means of deleting regions of the PAV genome, to provide
sites into which heterologous or homologous nucleotide sequences
encoding foreign genes or fragments thereof can be inserted to
generate porcine adenovirus recombinants. In preferred embodiments,
deletions are made in part or all of the nucleotide sequences of
the PAV E1, E3, or E4 regions and/or the region between E4 and the
right end of genome. E1 gene region deletions are described in
Example 3 and Example 10. E3 deletion and insertion of heterologous
sequence in the E3 region are described in Example 4 and 5; and
insertion of a heterologous sequence between the E4 region and the
right end of the PAV genome, as well as expression of the inserted
sequence, is described in Example 6, infra. E4 region deletions are
shown in Example 14.
[0074] In another embodiment, the invention identifies and provides
additional regions of the PAV genome (and fragments thereof)
suitable for insertion of heterologous or homologous nucleotide
sequences encoding foreign genes or fragments thereof to generate
PAV recombinants. These regions include nucleotides 145-13,555;
15,284-19,035; 22,677-24,055; 26,573-27,088; and 31,149-34,094 and
comprise the E2 region, the late region, and genes encoding the
pIX, DBP, pTP, pol, IVa2, 52K, IIIA, pIII, pVII, pV, pX, pVI, and
33K proteins. These regions of the PAV genome can be used, among
other things, for insertion of foreign sequences, for provision of
DNA control sequences including transcriptional and translational
regulatory sequences, or for diagnostic purposes to detect the
presence, in a biological sample, of viral nucleic acids and/or
proteins encoded by these regions. Example 7, infra, describes
procedures for constructing insertions in these regions.
[0075] One or more heterologous sequences can be inserted into one
or more regions of the PAV genome to generate a recombinant PAV
vector, limited only by the insertion capacity of the PAV genome
and ability of the recombinant PAV vector to express the inserted
heterologous sequences. In general, adenovirus genomes can accept
inserts of approximately 5% of genome length and remain capable of
being packaged into virus particles. The insertion capacity can be
increased by deletion of non-essential regions and/or deletion of
essential regions whose function is provided by a helper cell line.
In some examples, E40RF1-ORF2 and ORF4-ORF7 non essential regions
and E1B.sup.small are deleted to provide additional insertion
capacity.
[0076] In one embodiment of the invention, insertion can be
achieved by constructing a plasmid containing the region of the PAV
genome into which insertion is desired. The plasmid is then
digested with a restriction enzyme having a recognition sequence in
the PAV portion of the plasmid, and a heterologous sequence is
inserted at the site of restriction digestion. The plasmid,
containing a portion of the PAV genome with an inserted
heterologous sequence, in co-transformed, along with a plasmid
(such as pPAV-200) containing a full-length PAV genome, into a
bacterial cell (such as, for example, E. coli), wherein homologous
recombination between the plasmids generates a full-length PAV
genome containing inserted heterologous sequences.
[0077] Deletion of PAV sequences, to provide a site for insertion
of heterologous sequences or to provide additional capacity for
insertion at a different site, can be accomplished by methods
well-known to those of skill in the art. For example, for PAV
sequences cloned in a plasmid, digestion with one or more
restriction enzymes (with at least one recognition sequence in the
PAV insert) followed by ligation will, in some cases, result in
deletion of sequences between the restriction enzyme recognition
sites. Alternatively, digestion at a single restriction enzyme
recognition site within the PAV insert, followed by exonuclease
treatment, followed by ligation will result in deletion of PAV
sequences adjacent to the restriction site. A plasmid containing
one or more portions of the PAV genome with one or more deletions,
constructed as described above, can be co-transfected into a
bacterial cell along with a plasmid containing a full-length PAV
genome to generate, by homologous recombination, a plasmid
containing a PAV genome with a deletion at a specific site. PAV
virions containing the deletion can then be obtained by
transfection of mammalian cells (such as ST or VIDO R1 cells) with
the plasmid containing a PAV genome with a deletion at a specific
site.
[0078] Expression of an inserted sequence in a recombinant PAV
vector will depend on the insertion site. Accordingly, preferred
insertion sites are adjacent to and downstream (in the
transcriptional sense) of PAV promoters. The transcriptional map of
PAV, as disclosed herein, provides the locations of PAV promoters.
Locations of restriction enzyme recognition sequences downstream of
PAV promoters, for use as insertion sites, can be easily determined
by one of skill in the art from the PAV nucleotide sequence
provided herein. Alternatively, various in vitro techniques can be
used for insertion of a restriction enzyme recognition sequence at
a particular site, or for insertion of heterologous sequences at a
site that does not contain a restriction enzyme recognition
sequence. Such methods include, but are not limited to,
oligonucleotide-mediated heteroduplex formation for insertion of
one or more restriction enzyme recognition sequences (see, for
example, Zoller et al. (1982) Nucleic Acids Res. 10:6487-6500;
Brennan et al. (1990) Roux's Arch. Dev. Biol. 199:89-96; and Kunkel
et al. (1987) Meth. Enzymology 154:367-382) and PCR-mediated
methods for insertion of longer sequences. See, for example, Zheng
et al. (1994) Virus Research 31:163-186.
[0079] It is also possible to obtain expression of a heterologous
sequence inserted at a site that is not downstream from a PAV
promoter, if the heterologous sequence additionally comprises
transcriptional regulatory sequences that are active in eukaryotic
cells. Such transcriptional regulatory sequences can include
cellular promoters such as, for example, the bovine hsp70 promoter
and viral promoters such as, for example, herpesvirus, adenovirus
and papovavirus promoters and DNA copies of retroviral long
terminal repeat (LTR) sequences.
[0080] In another embodiment, homologous recombination in a
procaryotic cell can be used to generate a cloned PAV genome; and
the cloned PAV-3 genome can be propagated as a plasmid. Infectious
virus can be obtained by transfection of mammalian cells with the
cloned PAV genome rescued from plasmid-containing cells. Example 2,
infra describes construction of an infectious plasmid containing a
PAV-3 genome.
[0081] The invention provides PAV regulatory sequences which can be
used to regulate the expression of heterologous genes. A regulatory
sequence can be, for example, a transcriptional regulatory
sequence, a promoter, an enhancer, an upstream regulatory domain, a
splicing signal, a polyadenylation signal, a transcriptional
termination sequence, a translational regulatory sequence, a
ribosome binding site and a translational termination sequence.
[0082] Therapeutic Genes and Polypeptides
[0083] The PAV vectors of the invention can be used for the
expression of, production of, therapeutic polypeptides in
applications such as in vitro polypeptide production, vaccine
production, nucleic acid immunization and gene delivery, for
example. The PAV vectors of the present invention can be used to
produce polypeptides, of therapeutic or diagnostic value.
Therapeutic polypeptides comprise any polypeptide sequence with
therapeutic and/or diagnostic value and include, but are not
limited to, coagulation factors, growth hormones, cytokines,
lymphokines, tumor-suppressing polypeptides, cell receptors,
ligands for cell receptors, protease inhibitors, antibodies,
toxins, immunotoxins, dystrophins, cystic fibrosis transmembrane
conductance regulator (CFTR) and immunogenic polypeptides.
[0084] In some examples, PAV vectors will comprise heterologous
sequences encoding protective determinants of various pathogens of
mammals such as for example, humans or swine, for use in subunit
vaccines and nucleic acid immunization. Representative swine
pathogen antigens include, but are not limited to, pseudorabies
virus (PRV) gp50; transmissible gastroenteritis virus (TGEV) S
gene; porcine rotavirus VP7 and VP8 genes; genes of porcine
respiratory and reproductive syndrome virus (PRRS), in particular
ORFs 3, 4 and 5; genes of porcine epidemic diarrhea virus; genes of
hog cholera virus, genes of porcine parvovirus, and genes of
porcine influenza virus. Representative human pathogens include,
but are not limited to, HIV virus and Hepatitis virus.
[0085] Various foreign genes or nucleotide sequences or coding
sequences (prokaryotic, and eukaryotic) can be inserted into a PAV
vector, in accordance with the present invention, particularly to
provide protection against a wide range of diseases for use in
mammals including humans and swine. Many such genes are already
known in the art; the problem heretofore having been to provide a
safe, convenient and effective vaccine vector for the genes or
sequences.
[0086] A heterologous (i.e., foreign) nucleotide sequence can
consist of one or more gene(s) of interest, and preferably of
therapeutic interest. In the context of the present invention, a
gene of interest can code either for an antisense RNA, a ribozyme
or for an mRNA which will then be translated into a protein of
interest. A gene of interest can be of genomic type, of
complementary DNA (cDNA) type or of mixed type (minigene, in which
at least one intron is deleted). It can code for a mature protein,
a precursor of a mature protein, in particular a precursor intended
to be secreted and accordingly comprising a signal peptide, a
chimeric protein originating from the fusion of sequences of
diverse origins, or a mutant of a natural protein displaying
improved or modified biological properties. Such a mutant can be
obtained by deletion, substitution and/or addition of one or more
nucleotide(s) of the gene coding for the natural protein, or any
other type of change in the sequence encoding the natural protein,
such as, for example, transposition or inversion.
[0087] A gene of interest can be placed under the control of
regulatory sequences suitable for its expression in a host cell.
Suitable regulatory sequences are understood to mean the set of
elements needed for transcription of a gene into RNA (ribozyme,
antisense RNA or mRNA), for processing of RNA, and for the
translation of an mRNA into protein. Among the elements needed for
transcription, the promoter assumes special importance. It can be a
constitutive promoter or a regulatable promoter, and can be
isolated from any gene of eukaryotic, prokaryotic or viral origin,
and even adenoviral origin. Alternatively, it can be the natural
promoter of the gene of interest. Generally speaking, a promoter
used in the present invention can be chosen to contain
cell-specific regulatory sequences, or modified to contain such
sequences. For example, a gene of interest for use in the present
invention is placed under the control of an immunoglobulin gene
promoter when it is desired to target its expression to lymphocytic
host cells. There may also be mentioned the HSV-1 TK (herpesvirus
type 1 thymidine kinase) gene promoter, the adenoviral MLP (major
late promoter), in particular of human adenovirus type 2, the RSV
(Rous Sarcoma Virus) LTR (long terminal repeat), the CMV
(Cytomegalovirus) early promoter, and the PGK (phosphoglycerate
kinase) gene promoter, for example, permitting expression in a
large number of cell types.
[0088] Alternatively, targeting of a recombinant PAV vector to a
particular cell type can be achieved by constructing recombinant
hexon and/or fiber genes. The protein products of these genes are
involved in host cell recognition; therefore, the genes can be
modified to contain peptide sequences that will allow the virus to
recognize alternative host cells.
[0089] Among genes of interest which are useful in the context of
the present invention, there may be mentioned:
[0090] genes coding for cytokines such as interferons and
interleukins;
[0091] genes encoding lymphokines;
[0092] genes coding for membrane receptors such as the receptors
recognized by pathogenic organisms (viruses, bacteria or
parasites), preferably by the HIV virus (human immunodeficiency
virus);
[0093] genes coding for coagulation factors such as factor VIII and
factor IX;
[0094] genes coding for dystrophins;
[0095] genes coding for insulin;
[0096] genes coding for proteins participating directly or
indirectly in cellular ion channels, such as the CFTR (cystic
fibrosis transmembrane conductance regulator) protein;
[0097] genes coding for antisense RNAs, or proteins capable of
inhibiting the activity of a protein produced by a pathogenic gene
which is present in the genome of a pathogenic organism, or
proteins (or genes encoding them) capable of inhibiting the
activity of a cellular gene whose expression is deregulated, for
example an oncogene;
[0098] genes coding for a protein inhibiting an enzyme activity,
such as .alpha..sub.1-antitrypsin or a viral protease inhibitor,
for example;
[0099] genes coding for variants of pathogenic proteins which have
been mutated so as to impair their biological function, such as,
for example, trans-dominant variants of the tat protein of the HIV
virus which are capable of competing with the natural protein for
binding to the target sequence, thereby preventing the activation
of HIV;
[0100] genes coding for antigenic epitopes in order to increase the
host cell's immunity;
[0101] genes coding for major histocompatibility complex classes I
and II proteins, as well as the genes coding for the proteins which
are inducers of these genes;
[0102] genes coding for antibodies;
[0103] genes coding for immunotoxins;
[0104] genes encoding toxins;
[0105] genes encoding growth factors or growth hormones;
[0106] genes encoding cell receptors and their ligands;
[0107] genes encoding tumor suppressors;
[0108] genes coding for cellular enzymes or those produced by
pathogenic organisms; and
[0109] suicide genes. The HS V-1 TK suicide gene may be mentioned
as an example. This viral TK enzyme displays markedly greater
affinity compared to the cellular TK enzyme for certain nucleoside
analogues (such as acyclovir or gancyclovir). It converts them to
monophosphorylated molecules, which can themselves be converted by
cellular enzymes to nucleotide precursors, which are toxic. These
nucleotide analogues can be incorporated into replicating DNA
molecules, hence incorporation occurs chiefly in the DNA of
dividing cells. This incorporation can result in specific
destruction of dividing cells such as cancer cells.
[0110] This list is not restrictive, and any other gene of interest
can be used in the context of the present invention. In some cases
the gene for a particular antigen can contain a large number of
introns or can be from an RNA virus, in these cases a complementary
DNA copy (cDNA) can be used. It is also possible that only
fragments of nucleotide sequences of genes can be used (where these
are sufficient to generate a protective immune response or a
specific biological effect) rather than the complete sequence as
found in the wild-type organism. Where available, synthetic genes
or fragments thereof can also be used. However, the present
invention can be used with a wide variety of genes, fragments and
the like, and is not limited to those set out above.
[0111] Recombinant PAV vectors can be used to express antigens for
provision of, for example, subunit vaccines for use in mammals
including humans and swine. Antigens used in the present invention
can be either native or recombinant antigenic polypeptides or
fragments. They can be partial sequences, full-length sequences, or
even fusions (e.g., having appropriate leader sequences for the
recombinant host, or with an additional antigen sequence for
another pathogen). The preferred antigenic polypeptide to be
expressed by the virus systems of the present invention contain
full-length (or near full-length) sequences encoding antigens.
Alternatively, shorter sequences that are antigenic (i.e., encode
one or more epitopes) can be used. The shorter sequence can encode
a "neutralizing epitope," which is defined as an epitope capable of
eliciting antibodies that neutralize virus infectivity in an in
vitro assay. Preferably the peptide should encode a "protective
epitope" that is capable of raising in the host a "protective
immune response;" i.e., a humoral (i.e. antibody-mediated),
cell-mediated, and/or mucosal immune response that protects an
immunized host from infection.
[0112] The antigens used in the present invention, particularly
when comprised of short oligopeptides, can be conjugated to a
vaccine carrier. Vaccine carriers are well known in the art: for
example, bovine serum albumin (BSA), human serum albumin (HSA) and
keyhole limpet hemocyanin (KLH). A preferred carrier protein,
rotavirus VP6, is disclosed in EPO Pub. No. 0259149, the disclosure
of which is incorporated by reference herein.
[0113] Genes for desired antigens or coding sequences thereof which
can be inserted include those of organisms which cause disease in
mammals, particularly porcine pathogens such as pseudorabies virus
(PRV), transmissible gastroenteritis virus (TGEV), porcine
rotavirus, porcine respiratory and reproductive syndrome virus
(PRRS), porcine epidemic diarrhea virus (PEDV), hog cholera virus
(HCV), porcine parvovirus and the like. Genes encoding antigens of
human pathogens, such as HIV and Hepatitis are also useful in the
practice of the invention.
[0114] Therapeutic Applications
[0115] With the recombinant viruses of the present invention, it is
possible to elicit an immune response against disease antigens
and/or provide protection against a wide variety of diseases
affecting swine, cattle, humans and other mammals. Any of the
recombinant antigenic determinants or recombinant live viruses of
the invention can be formulated and used in substantially the same
manner as described for the antigenic determinant vaccines or live
vaccine vectors.
[0116] The present invention also includes pharmaceutical
compositions comprising a therapeutically effective amount of a
recombinant vector, recombinant virus or recombinant protein,
prepared according to the methods of the invention, in combination
with a pharmaceutically acceptable vehicle and/or an adjuvant. Such
a pharmaceutical composition can be prepared and dosages determined
according to techniques that are well-known in the art. The
pharmaceutical compositions of the invention can be administered by
any known administration route including, but not limited to,
systemically (for example, intravenously, intratracheally,
intraperitoneally, intranasally, parenterally, enterically,
intramuscularly, subcutaneously, intratumorally or intracranially)
or by aerosolization or intrapulmonary instillation. Administration
can take place in a single dose or in doses repeated one or more
times after certain time intervals. The appropriate administration
route and dosage will vary in accordance with the situation (for
example, the individual being treated, the disorder to be treated
or the gene or polypeptide of interest), but can be determined by
one of skill in the art.
[0117] The vaccines of the invention carrying foreign genes or
fragments can be orally administered in a suitable oral carrier,
such as in an enteric-coated dosage form. Oral formulations include
such normally-employed excipients as, for example, pharmaceutical
grades of mannitol, lactose, starch, magnesium stearate, sodium
saccharin cellulose, magnesium carbonate, and the like. Oral
vaccine compositions may be taken in the form of solutions,
suspensions, tablets, pills, capsules, sustained release
formulations, or powders, containing from about 10% to about 95% of
the active ingredient, preferably about 25% to about 70%. An oral
vaccine may be preferable to raise mucosal immunity (which plays an
important role in protection against pathogens infecting the
gastrointestinal tract) in combination with systemic immunity.
[0118] In addition, the vaccine can be formulated into a
suppository. For suppositories, the vaccine composition will
include traditional binders and carriers, such as polyalkaline
glycols or triglycerides. Such suppositories may be formed from
mixtures containing the active ingredient in the range of about
0.5% to about 10% (w/w), preferably about 1% to about 2%.
[0119] Protocols for administering to animals the vaccine
composition(s) of the present invention are within the skill of the
art in view of the present disclosure. Those skilled in the art
will select a concentration of the vaccine composition in a dose
effective to elicit antibody, cell-mediated and/or mucosal immune
responses to the antigenic fragment. Within wide limits, the dosage
is not believed to be critical. Typically, the vaccine composition
is administered in a manner which will deliver between about 1 to
about 1,000 micrograms of the subunit antigen in a convenient
volume of vehicle, e.g., about 1-10 ml. Preferably, the dosage in a
single immunization will deliver from about 1 to about 500
micrograms of subunit antigen, more preferably about 5-10 to about
100-200 micrograms (e.g., 5-200 micrograms).
[0120] The timing of administration may also be important. For
example, a primary inoculation preferably may be followed by
subsequent booster inoculations, for example, several weeks to
several months after the initial immunization, if needed. To insure
sustained high levels of protection against disease, it may be
helpful to re-administer booster immunizations at regular
intervals, for example once every several years. Alternatively, an
initial dose may be administered orally followed by later
inoculations, or vice versa. Preferred vaccination protocols can be
established through routine vaccination protocol experiments.
[0121] The dosage for all routes of administration of in vivo
recombinant virus vaccine depends on various factors including, the
size of patient, nature of infection against which protection is
needed, carrier and the like and can readily be determined by those
of skill in the art. By way of non-limiting example, a dosage of
between approximately 10.sup.3 pfu and 10.sup.8 pfu can be used. As
with in vitro subunit vaccines, additional dosages can be given as
determined by the clinical factors involved.
[0122] A problem that has beset the use of adenovirus vectors for
immunization and gene delivery in humans is the rapid development
of an immunological response (or indeed in some cases existing
immunity) to human adenoviruses (HAVs). Recombinant PAV vectors are
likely to be less immunogenic in humans and, for this and other
reasons, will be useful either as a substitute for HAV vectors or
in combination with HAV vectors. For example, an initial
immunization with a HAV vector can be followed by booster
immunizations using PAV vectors; alternatively, initial
immunization with a recombinant PAV vector can be followed by
booster immunizations with HAV and/or PAV vectors. As shown herein
in Examples 14 and 15, PAV can infect a variety of human cell
lines.
[0123] The presence of low levels of helper-independent vectors in
the batches of helper-dependent human adenoviruses that are grown
in complementing human cell lines has been reported. Fallaux et
al., (1998) supra. This occurs as a result of recombination events
between the viral DNA and the integrated adenoviral sequences
present in the complementing cell line. Hehir et al. (1996) J.
Virol. 70:8459-8467. This type of contamination constitutes a
safety risk, which could result in the replication and spread of
the virus. Complete elimination of helper-dependent adenoviruses in
the batches of helper-dependent vectors can be achieved using two
approaches. The first is by developing new helper cell lines and
matched vectors that do not share any common sequences. Fallaux et
al. (1998) supra. The second approach is to take advantage of
possible cross-complementation between two distantly related
adenoviruses such as HAV-5 and PAV-3. VIDO R1 cells contain the E1
coding sequences of HAV-5. Although there is no significant
homology between the E1 regions of HAV-5 and PAV-3 at the
nucleotide sequence level, the proteins produced from the region
can complement each others' function(s). Thus, the problem of
helper-independent vector generation by homologous recombination is
eliminated when VIDO R1 cells are used for the propagation of
recombinant PAV-3.
[0124] The invention also encompasses a method of treatment,
according to which a therapeutically effective amount of a PAV
vector, recombinant PAV, or host cell of the invention is
administered to a mammalian subject requiring treatment. The
finding that PAV-3 was effective in entering canine, sheep and
bovine cells in which it does not replicate or replicates poorly is
an important observation. See Example 8, infra. This may have
implications in designing PAV-3 vectors for vaccination in these
and other animal species. As shown herein, PAV is able to replicate
in a number of mammalian cell lines.
[0125] Recombinant PAV vectors can be used for regulated expression
of foreign polypeptides encoded by heterologous nucleotide
sequences. Standard conditions of cell culture, such as are known
to those of skill in the art, will allow maximal expression of
recombinant polypeptides. They can be used, in addition, for
regulated expression of RNAs encoded by heterologous nucleotide
sequences, as in, for example, antisense applications and
expression of ribozymes.
[0126] When the heterologous sequences encode an antigenic
polypeptide, PAV vectors comprising insertions of heterologous
nucleotide sequences can be used to provide large quantities of
antigen which are useful, in turn, for the preparation of
antibodies. Methods for preparation of antibodies are well-known to
those of skill in the art. Briefly, an animal (such as a rabbit) is
given an initial subcutaneous injection of antigen plus Freund's
complete adjuvant. One to two subsequent injections of antigen plus
Freund's incomplete adjuvant are given at approximately 3 week
intervals. Approximately 10 days after the final injection, serum
is collected and tested for the presence of specific antibody by
ELISA, Western Blot, immunoprecipitation, or any other
immunological assay known to one of skill in the art.
[0127] Adenovirus E1 gene products transactivate many cellular
genes; therefore, cell lines which constitutively express E1
proteins can express cellular polypeptides at a higher levels than
other cell lines. The recombinant mammalian, particularly porcine,
cell lines of the invention can be used to prepare and isolate
polypeptides, including those such as (a) proteins associated with
adenovirus E1A proteins: e.g. p300, retinoblastoma (Rb) protein,
cyclins, kinases and the like; (b) proteins associated with
adenovirus E1B protein: e.g. p53 and the like; growth factors, such
as epidermal growth factor (EGF), transforming growth factor (TGF)
and the like; (d) receptors such as epidermal growth factor
receptor (EGF-R), fibroblast growth factor receptor (FGF-R), tumor
necrosis factor receptor (TNF-R), insulin-like growth factor
receptor (IGF-R), major histocompatibility complex class I receptor
and the like; (e) proteins encoded by proto-oncogenes such as
protein kinases (tyrosine-specific protein kinases and protein
kinases specific for serine or threonine), p21 proteins (guanine
nucleotide-binding proteins with GTPase activity) and the like; (f)
other cellular proteins such as actins, collagens, fibronectins,
integrins, phosphoproteins, proteoglycans, histones and the like,
and (g) proteins involved in regulation of transcription such as
TATA-box-binding protein (TBP), TBP-associated factors (TAFs), SpI
binding protein and the like.
[0128] Gene Delivery
[0129] The invention also includes a method for delivering a gene
to a mammal, such as a porcine, human or other mammal in need
thereof, to control a gene deficiency. In one embodiment, the
method comprises administering to said mammal a live recombinant
porcine adenovirus containing a heterologous nucleotide sequence
encoding a non-defective form of said gene under conditions wherein
the recombinant virus vector genome is incorporated into said
mammalian genome or is maintained independently and
extrachromosomally to provide expression of the required gene in
the target organ or tissue. These kinds of techniques are currently
being used by those of skill in the art to replace a defective gene
or portion thereof. Examples of foreign genes, heterologous
nucleotide sequences, or portions thereof that can be incorporated
for use in gene therapy include, but are not limited to, cystic
fibrosis transmembrane conductance regulator gene, human
minidystrophin gene, alpha-1-antitrypsin gene and the like.
[0130] In particular, the practice of the present invention in
regard to gene delivery in humans is intended for the prevention or
treatment of diseases including, but not limited to, genetic
diseases (for example, hemophilia, thalassemias, emphysema,
Gaucher's disease, cystic fibrosis, Duchenne muscular dystrophy,
Duchenne's or Becker's myopathy, etc.), cancers, viral diseases
(for example, AIDS, herpesvirus infection, cytomegalovirus
infection and papillomavirus infection) and the like. For the
purposes of the present invention, the vectors, cells and viral
particles prepared by the methods of the invention may be
introduced into a subject either ex vivo, (i.e., in a cell or cells
removed from the patient) or directly in vivo into the body to be
treated. Preferably, the host cell is a human cell and, more
preferably, is a lung, fibroblast, muscle, liver or lymphocytic
cell or a cell of the hematopoietic lineage.
[0131] Diagnostic Applications
[0132] The PAV genome, or any subregion of the PAV genome, is
suitable for use as a nucleic acid probe, to test for the presence
of PAV nucleic acid in a subject or a biological sample. The
presence of viral nucleic acids can be detected by techniques known
to one of skill in the art including, but not limited to,
hybridization assays, polymerase chain reaction, and other types of
amplification reactions. Suitable labels and hybridization
techniques are well-known to those of skill in the art. See, for
example, Kessler (ed.), Nonradioactive Labeling and Detection of
Biomolecules, Springer-Verlag, Berlin, 1992; Kricka (ed.)
Nonisotopic DNA Probe Techniques, Academic Press, San Diego, 1992;
Howard (ed.) Methods in Nonradioactive Detection, Appleton &
Lange, Norwalk, 1993; Ausubel et al., supra; and Sambrook et al.,
supra. Diagnostic kits comprising the nucleotide sequences of the
invention can also contain reagents for cell disruption and nucleic
acid purification, as well as buffers and solvents for the
formation, selection and detection of hybrids.
[0133] Regions of the PAV genome can be inserted into any
expression vector known in the art and expressed to provide, for
example, vaccine formulations, protein for immunization, etc. The
amino acid sequence of any PAV protein can be determined by one of
skill in the art from the nucleotide sequences disclosed herein.
PAV proteins can be used for diagnostic purposes, for example, to
detect the presence of PAV antigens. Methods for detection of
proteins are well-known to those of skill in the art and include,
but are not limited to, various types of direct and competitive
immunoassays, ELISA, Western blotting, enzymatic assay,
immunohistochemistry, etc. See, for example, Harlow & Lane
(eds.): Antibodies, A Laboratory Manual, Cold Spring Harbor Press,
New York, 1988. Diagnostic kits comprising PAV polypeptides or
amino acid sequences can also comprise reagents for protein
isolation and for the formation, isolation, purification and/or
detection of immune complexes.
EXAMPLES
Materials and Methods
Virus and Viral DNA.
[0134] The 6618 strain of PAV-3 was propagated in the swine testis
(ST) cell line and in E1-transformed porcine retinal cells (VIDO
R1, see below). Porcine embryonic retinal cells were obtained from
the eyeballs of piglets delivered by caesarian section two weeks
before the parturition date. Uninfected cells were grown in MEM
supplemented with 10% fetal bovine serum (FBS). MEM with 2% FBS was
used for maintenance of infected cells. Viral DNA was extracted
either from infected cell monolayers by the method of Hirt (1967)
J. Mol. Biol. 26:365-369, or from purified virions as described by
Graham et al. (1991) in "Methods in Molecular Biology" Vol. 7, Gene
transfer and expression protocols, ed. E.J. Murray, Humana Press,
Clifton, N.J., pp. 109-128.
[0135] Plasmids and Genomic DNA Sequencing.
[0136] Selected restriction enzyme fragments of PAV-3 DNA were
cloned into pGEM-3Z and pGEM-7Zf(+) plasmids (Promega). Nucleotide
sequences were determined on both strands of the genome by the
dideoxy chain-termination method using Sequenase.RTM. enzyme (U.S.
Biochemicals) and the dye-terminator method with an Applied
Biosystems (Foster City, Calif.) DNA sequencer.
[0137] cDNA Library.
[0138] A cDNA library was generated from polyadenylated RNA
extracted from PAV-3 infected ST cells at 12 h and 24 h post
infection. Double stranded cDNAs were made with reagents from
Stratagene and cloned into Lambda ZAP vector. Plaques which
hybridized to specific restriction enzyme fragments of PAV-3 DNA
were plaque purified twice. Plasmids containing cDNAs were excised
from the Lambda ZAP vector according to the manufacturer's
protocol. The resulting plasmid clones were characterized by
restriction endonuclease analysis and by sequencing of both ends of
the cDNA insert with T3- and T7-specific primers. Selected clones
were sequenced with internal primers. cDNA sequences were aligned
with genomic sequences to determine the transcription map.
[0139] Viral Transcript Mapping by Nuclease Protection
[0140] Transcript mapping was conducted according to the method of
Berk et al. (1977) Cell 12:721-732.
Example 1
Development of an E1-Complementing Helper Cell Line (VIDO R1)
[0141] Primary cultures of porcine embryonic retina cells were
transfected with 10 .mu.g of plasmid pTG 4671 (Transgene,
Strasbourg, France) by the calcium phosphate technique. The pTG
4671 plasmid contains the entire E1A and EIB sequences (nts
505-4034) of HAV-5, along with the puromycin acetyltransferase gene
as a selectable marker. In this plasmid, the E1 region is under the
control of the constitutive promoter from the mouse
phosphoglycerate kinase gene, and the puromycin acetyltransferase
gene is controlled by the constitutive SV40 early promoter.
Transformed cells were selected by three passages in medium
containing 7 .mu.g/ml puromycin, identified based on change in
their morphology from single foci (i.e., loss of contact
inhibition), and subjected to single cell cloning. The established
cell line was first tested for its ability to support the growth of
E1 deletion mutants of HAV-5. Subsequently the cell line was
further investigated for the presence of E1 sequences in the genome
by PCR, expression of the E1A and E1B proteins by Western blot, and
doubling time under cell culture conditions. E1 sequences were
detected, and production of E1A and E1B proteins was demonstrated
by immunoprecipitation (FIG. 3). Doubling time was shorter, when
compared to that of the parent cell line. Example 3, infra, shows
that this cell line is capable of complementing a PAV E1A deletion
mutant.
[0142] To assess the stability of E1 expression, VIDO R1 cells were
cultured through more than 50 passages (split 1:3 twice weekly) and
tested for their ability to support the replication of E1-deleted
HAV-5. Expression of the E1A and E1B proteins at regular intervals
was also monitored by Western blot. The results indicated that the
VIDO R1 line retained the ability to support the growth of
E1-deleted virus and expressed similar levels of E1 proteins during
more than 50 passages in culture. Therefore, VIDO R1 can be
considered to be an established cell line.
Example 2
Construction of a Full-Length Infectious Clone of PAV-3
[0143] A plasmid clone containing a full-length copy of the PAV-3
genome (pPAV-200) was generated by first constructing a plasmid
containing left- and right-end sequences of PAV-3, with the PAV-3
sequences bordered by Pa c sites and separated by a PstI
restriction site (pPAV-100), then allowing recombination between
PstI-digested pPAV-100 and an intact PAV-3 genome. Left- and
right-end sequences for insertion into pPAV-100 were produced by
PCR amplification, as follows.
[0144] The plasmid p3SB (Reddy et al., 1993, Intervirology
36:161-168), containing the left end of PAV-3 genome (position
1-8870) was used for amplification of the first 433 bp of the PAV-3
genome by PCR. Amplification primers were oligonucleotides 1
(5'-GCGGATCCTTAATTAACATCATCAATAATATACCGCACACTTTT-3') (SEQ ID NO.:
2) and 2 (5'-CACCTGCAGATACACCCACACACGTCATCTCG-3') (SEQ ID NO.: 3).
In the sequences shown here, adenoviral sequences are shown in
bold/underlined and engineered restriction enzyme sites are
italicized.
[0145] For amplification of sequences at the right end of the PAV-3
genome, the plasmid p3SA (Reddy et al., 1993, supra) was used. This
plasmid was used as template in PCR for amplification of the
terminal 573 bp of the genome using oligonucleotide 1 (above) and
oligonucleotide 3
TABLE-US-00003 (SEQ ID NO.: 4)
(5'-CACCTGCAGCCTCCTGAGTGTGAAGAGTGTCC-3').
The primers were designed based on the nucleotide sequence
information described elsewhere (Reddy et al., 1995c, supra; and
Reddy et al., 1997, supra).
[0146] For construction of pPAV-100, the PCR product obtained with
oligonucleotides 1 and 2 was digested with BamHI and PstI
restriction enzymes and the PCR product obtained using primers 1
and 3 was digested with PstI and Pa c enzymes. Modified bacterial
plasmid pPolyIIsn14 was digested with BamHI and Pa c enzymes. This
plasmid was used based on its suitability for homologous
recombination in E. coli. The two PCR products described above were
cloned into pPolyIIsn14 by three way ligation to generate the
plasmid pPAV-100 which carries both termini of PAV-3, separated by
a PstI site and bordered by Pa c restriction enzyme sites.
[0147] Plasmid pPAV-200, which contains a full length PAV-3 genome,
was generated by co-transformation of E. coli BJ 5183 recBC sbcBC
(Hanahan, 1983, J. Mol. Biol. 166:557-580) with PstI-linearized
pPAV-100 and the genomic DNA of PAV-3. Extensive restriction enzyme
analysis of pPAV-200 indicated that it had the structure expected
of a full-length PAV-3 insert, and that no unexpected
rearrangements had occurred during recombination in E. coli.
[0148] The infectivity of pPAV-200 was demonstrated by lipofectin
transfection (Life Technologies, Gaithersburg, Md.) of ST cells
following PacI enzyme digestion of the plasmid to release the viral
genome from the plasmid. Viral plaques were evident 7 days
following transfection, and titers were equivalent to, or higher
than, those obtained after infection with wild-type PAV. The
plaques were amplified and the viral DNA was extracted and analyzed
by restriction enzyme digestion. The viral DNA obtained by cleavage
of pPAV-200 with Pa c contained an extra 3 bases at each end; but
these extra bases did not substantially reduce the infectivity of
the PAV genome excised from pPAV-200. In addition, the
bacterial-derived genomes lacked the 55-kDa terminal protein that
is covalently linked to the 5' ends of adenoviral DNAs and which
enhances infectivity of viral DNA.
Example 3
Generation of E1 Deletion Mutants of PAV-3
[0149] A plasmid (pPAV-101) containing the left (nucleotides
1-2,130) and the right (nucleotides 32,660-34,094) terminal NcoI
fragments of the PAV-3 genome was constructed by digesting pPAV-200
with the enzyme NcoI (which has no recognition sites in the vector
backbone, but many sites in the PAV insert), gel-purifying the
appropriate fragment and self-ligating the ends. See FIG. 4. The
E1A sequences of pPAV-101, between nucleotides 407 and 1270 (PAV
genome numbering), were deleted by digestion of pPAV-101 with NotI
(recognition site at nucleotide 407) and AseI (recognition site at
1270), generation of blunt ends, and insertion of a double-stranded
oligonucleotide encoding a XbaI restriction site to create a
plasmid, pPAV-102, containing PAV left- and right-end sequences,
separated by a NcoI site, with a deletion of the E1A region and a
XbaI site at the site of the deletion. See FIG. 5. Plasmid
pPAV-201, containing a full-length PAV-3 genome minus E1A
sequences, was created by co-transformation of E. coli BJ 5183 with
NcoI linearized pPAV-102 and genomic PAV-3 DNA. The resulting
construct, when transfected into VIDO R1 cells following digestion
with PacI restriction enzyme, produced a virus that had a deletion
in the E1 region. In similar fashion, construction of a virus with
deletions in E1 and E3 was accomplished by transformation of BJ
5183 cells with NcoI linearized pPAV-102 and genomic PAV-3 DNA
containing an E3 deletion. These E1A deletion mutants did not grow
on either ST (swine testis) cells or fetal porcine retina cells and
could only be grown in the VIDO R1 cell line.
Example 4
Generation of E3 Inserts and Deletion Mutants
[0150] To systematically examine the extent of the E3 region that
could be deleted, a E3 transfer vector was constructed. The vector
(pPAV-301) contained a PAV-3 segment from nucleotides 26,716 to
31,064 with a green fluorescent protein (GFP) gene inserted into
the SnaBI site (located at nucleotide 28,702) in the same
orientation as E3. The GFP gene was obtained from the plasmid
pGreen Lantern-1.TM. (Life Technologies), by NotI digestion
followed by purification of a 732-nucleotide fragment. Similarly,
another construct was made with GFP cloned into the SacI site
located at nucleotide 27,789. KpnI-BamHI fragments encompassing the
modified E3 regions were then isolated from these E3 transfer
vectors and recombined in E coli with pPAV-200 that had been
linearized at nucleotide position 28,702 by SnaBI digestion. Virus
were obtained with a construct that had the GFP gene cloned into
the SnaBI site.
[0151] To delete the non-essential portion of E3 from the transfer
vector, a PCR approach was used. In this approach, the region of
the PAV genome between nucleotides 27,402 and 28,112 was amplified
using the following primers:
TABLE-US-00004 5'-GACTGACGCCGGCATGCAAT-3' SEQ ID NO: 5
5'-CGGATCCTGACGCTACGAGCGGTTGTA-3' SEQ ID NO: 6
In a second PCR reaction, the portion of the PAV genome between
nucleotides 28,709 and 29,859 was amplified using the following two
primers:
TABLE-US-00005 5'-CGGATCCATACGTACAGATGAAGTAGC-3' SEQ ID NO: 7
5'-TCTGACTGAAGCCGACCTGC-3' SEQ ID NO: 8
[0152] In the oligonucleotides designated SEQ ID NO: 6 and SEQ ID
NO: 7, a BamHI recognition sequence is indicated by underlining.
The template for amplification was a KpnI-BamHI fragment
encompassing nucleotides 26,716-31,063 of the PAV genome, inserted
into the plasmid pGEM3Z (Promega), and Pfu polymerase (Stratagene)
was used for amplification. The first PCR product (product of
amplification with SEQ ID NO: 5 and SEQ ID NO: 6) was digested with
BamHI and gel-purified. The second PCR product (product of
amplification with SEQ ID NO: 7 and SEQ ID NO: 8) was digested with
BamHI and SpeI and gel-purified. They were inserted into
SmaI/SpeI-digested pBlueScript II SK(+) (Stratagene) in a three-way
ligation reaction to generate pPAV-300. See FIG. 6. pPAV-300
contains the portion of the PAV-3 genome extending from nucleotides
27,402 to 29,859, with 594 base pairs (bp) between nucleotides
28,113 and 28,707 deleted from the E3 region. A virus with such a
deletion was constructed as follows. A SphI-SpeI fragment from
pPAV-300, containing part of the pVIII gene, a deleted E3 region,
and part of the fiber gene was isolated (see FIG. 6). This fragment
was co-transfected, with SnaBI-digested pPAV-200 (which contains a
full-length PAV-3 genome) into E. coli. Homologous recombination
generated a plasmid, pFPAV-300, containing a full-length PAV genome
with a deletion in the E3 region. pFPAV-300 was digested with PacI
and transfected into VIDO R1 cells (Example 1) to generate
recombinant virus with a deletion in the E3 region of the
genome.
Example 5
Construction of a PAV Recombinant with an Insertion of the PRV gp50
gene in the PAV E3 Region and Expression of the Inserted Gene
[0153] To construct a recombinant PAV expressing pseudorabies virus
(PRV) gp50, the PRV gp50 gene was inserted at the SnaBI site of
pPAV-300 to create plasmid pPAV-300-gp50. A SphI-SpeI fragment from
pPAV-300-gp50, containing part of the pVII gene, a deleted E3
region with the PRV gp50 gene inserted, and part of the fiber gene,
was purified and co-transfected, along with SnaBI-digested
pFPAV-300 (E3-deleted) into E. coli. In the bacterial cell,
homologous recombination generated pFPAV-300-gp50, a plasmid
containing a PAV genome with the PRV gp50 gene replacing a deleted
E3 region. Recombinant virus particles were obtained as described
in Example 4.
[0154] Expression of the inserted PRV gp50 was tested after
infection of VIDO R1 cells with the recombinant virus, by .sup.35S
labeling of infected cells (continuous label), followed by
immunoprecipitation with an anti-gp50 monoclonal antibody and gel
electrophoresis of the immunoprecipitate. FIG. 7 shows that large
amounts of gp50 are present by 12 hours after infection, and
expression of gp50 persists up to 24 hours after infection.
Example 6
Expression of the Chloramphenicol Acetyltransferase Gene from a
Region that Lies Between the Promoter of the E4 Region and the
Right ITR
[0155] The right terminal fragment of the PAV genome (encompassing
nucleotides 31,054-34,094) was obtained by XhoI digestion of
pPAV-200 and cloned between the XhoI and NotI sites of pPolyIIsn14.
A Chloramphenicol acetyltransferase (CAT) gene expression cassette,
in which the CAT gene was flanked by the SV40 early promoter and
the SV40 polyadenylation signal, was inserted, in both
orientations, into a unique HpaI site located between the E4 region
promoter and the right ITR, to generate plasmids pPAV-400A and
pPAV-400B. The modified terminal fragments were transferred into a
plasmid containing a full-length PAV-3 genome by homologous
recombination in E. coli between the isolated terminal fragments
and HpaI-digested pPAV-200. Recombinant viruses expressing CAT were
obtained following transfection of VIDO R1 cells with the plasmids.
PAV-CAT2 contained the CAT gene cassette in a leftward
transcriptional orientation (i.e., the same orientation as E4
region transcription), while, in PAV-CAT6, the CAT gene cassette
was in the rightward transcriptional orientation.
[0156] These recombinant viruses were tested for expression of CAT,
after infection of VIDO R1 cells, using a CAT Enzyme Assay System
from Promega, following the instructions provided by the supplier.
See, Cullen (1987) Meth. Enzymology 43:737; and Gorman et al.,
(1982) Mol. Cell. Biol. 2:1044. The results are shown in Table
3.
TABLE-US-00006 TABLE 3 CAT activity expressed by recombinant PAV
viruses Sample .sup.3H cpm Mock-infected 458 CAT positive control*
199,962 PAV-CAT2 153,444 PAV-CAT6 63,386 *the positive control
sample contained 0.1 Units of purified CAT.
[0157] These results show that recombinant PAV viruses, containing
an inserted gene, are viable and are capable of expressing the
inserted gene.
Example 7
Construction of Replication Defective PAV-3 Expressing GFP
[0158] A 2.3 kb fragment containing the CMV immediate early
promoter, the green fluorescent protein (GFP) gene and the bovine
growth hormone poly(A) signal was isolated by digesting pQBI 25
(Quantum Biotechnology) with BglII and DraIII followed by filling
the ends with T4 DNA polymerase. This fragment was inserted into
the SrjI site of pPAV-102 in both orientations to generate
pPAV-102GFP (FIG. 8). This plasmid, digested with Pa c and SmaI
enzymes, and the fragment containing part of the E1 sequence and
the GFP gene was gel purified. This fragment and the SrfI digested
pFPAV-201 were used to transform E. coli BJ 5183 to generate the
full-length clone containing GFP in the E1 region (pFPAV-201-GFP)
by homologous recombination. The recombinant virus, PAV3delE1E3.GFP
was generated following transfection of VIDO R1 cells with Pa c
restricted pFPAV-201-GFP that had the GFP transcription unit in the
opposite orientation to the E1. A similar virus with the GFP in the
same orientation as E1 could not be rescued from transfected cells.
Presence of the GFP gene in the viral genome was confirmed by
restriction enzyme analysis. The recombinant virus replicated in
VIDO R1 cells two logs less efficiently than the wild type
PAV-3.
Example 8
Virus Entry and Replication of PAV-3 in Human and Animal Cells
[0159] To initially characterize the host species restriction of
PAV in vitro, monolayers of II cell types from 6 different
mammalian species were infected with wild type PAV-3 or
PAV3del.E1E3.GFP. ST, VIDO R1 (porcine), 293, A549 (human), MDBK,
VIDO R2 (bovine, ATCC accession number PTA 156), C3HA (mouse), COS,
VERO (monkey), sheep skin fibroblasts or cotton rat lung cells were
incubated with 1 pfu/cell of wild type PAV-3 or helper-dependent
PAV-3 expressing GFP. The cells infected with wild type PAV were
harvested at 2 h and 3 days post-infection, subjected to two cycles
of freeze-thaw, and virus titers were determined on VIDO R1 cells.
Cells that were infected with the recombinant virus expressing GFP
were observed with the aid of a fluorescent microscope for green
fluorescence.
[0160] A ten-fold increase in virus titers in Vero and COS cells,
and a hundred-fold increase in cotton rat lung fibroblasts and VIDO
R2 cells, was noticed. No increase in the virus titers was observed
with 293, A549, MDBK, sheep skin fibroblasts, dog kidney and C3HA
cells. All of these cell types showed bright green fluorescence
when infected with PAV3delE1E3.GFP except human cells, which showed
a weak fluorescence. In addition, low levels of GFP expression were
achieved in human cells with recombinant PAV-3. These observations
suggest that virus entry into some human cells is limited and/or
the human cells are non-permissive for the replication of the
virus. These results also demonstrated that GFP was expressed by
the PAV-3 vector in cells which are semi-permissive (VERO, COS,
Cotton rat lung fibroblasts and VIDO R2), or non-permissive (Sheep
skin fibroblasts, MDBK and human cells) for virus replication.
Example 9
Insertions in the Regions of the PAV-3 Genome Defined by
Nucleotides 145-13,555; 15,284-19,035; 22,677-24,055;
26,573-27,088; and 31,149-34,094
[0161] Insertions are made by art-recognized techniques including,
but not limited to, restriction digestion, nuclease digestion,
ligation, kinase and phosphatase treatment, DNA polymerase
treatment, reverse transcriptase treatment, and chemical
oligonucleotide synthesis. Heterologous nucleic acid sequences of
interest are cloned into plasmid vectors containing portions of the
PAV genome (which may or may not contain deletions of PAV
sequences) such that the foreign sequences are flanked by sequences
having substantial homology to a region of the PAV genome into
which insertion is to be directed. Substantial homology refers to
homology sufficient to support homologous recombination. These
constructs are then introduced into host cells that are
co-transfected with PAV-3 DNA or a cloned PAV genome. During
infection, homologous recombination between these constructs and
PAV genomes will occur to generate recombinant PAV
genome-containing plasmids. Recombinant virus are obtained by
transfecting the recombinant PAV genome-containing plasmids into a
suitable mammalian host cell line. If the insertion occurs in an
essential region of the PAV genome, the recombinant PAV virus is
propagated in a helper cell line which supplies the viral function
that was lost due to the insertion.
Example 10
Analysis of Early Region 1 of Porcine Adenovirus
Materials and Methods
Cells and Viruses
[0162] VIDO R1 (Reddy et al., 1999(b), J. Gen. Virol. 80:2909-2916)
and Swine Testicular (ST) cells (ATCC Cat. No. CRL 1746) were grown
and maintained in minimum essential medium (MEM) supplemented with
10% fetal bovine serum (FBS). The PAV strains (wild-type PAV-3
strain 6618) were propagated and titrated in VIDO R1 cells (Reddy
et al., 1999(b), supra).
[0163] GST Fusion and Antibody Production
[0164] The plasmid pE1A was created by amplifying part of E1A (nt
556 to 1222) by PCR and ligating in-frame to glutathione
S-transferase (GST) gene in plasmid pGEX-5X-3. To create plasmid
pE1Bs, part of E1B.sup.small ORF (nt 1470 to 2070) was amplified by
PCR and ligated in-frame to the GST gene in plasmid pGEX-5X-3. The
plasmid pE1B1 was created by amplifying complete E1B.sup.large ORF
(nt 1831-3250) by PCR and ligated in-frame to the GST gene in
plasmid pGEX-5X-3. The junctions of the sequences encoding GST-E1A,
GST-E1B.sup.small or GST-E1B.sup.large were sequenced to ensure
that the coding domains are in frame. The competent Escherichia
coli strain BL121 was transformed with pE1A, pE1Bs or pE1B1
plasmids. The fusion protein(s) were induced by addition of 0.1 M
isopropyl-.beta.-.sub.D-thiogalactoside and purified using sodium
dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis (PAGE).
Rabbits were immunized subcutaneously with 300 ug of gel purified
GST-E1A, GST-E1B.sup.small or GST-E1B.sup.large fusion proteins in
Freund's complete followed by three injections in Freund's
incomplete adjuvant at 4-weeks interval.
[0165] In vitro Transcription and Translation
[0166] The complete coding regions of E1A, E1B.sup.small and
E1B.sup.large were individually cloned into the SmaI site of
plasmid pSP64 polyA creating plasmid pSP64-PE1A, pSP64-PE1Bs and
pSP64-PE1B1 respectively. The plasmid DNAs were transcribed and
translated in vitro by using a rabbit reticulocyte lysate coupled
transcription translation system in the presence of 50 .mu.Ci of
[.sup.35S]-methionine. The in vitro translated proteins were
analyzed with or without immunoprecipitation with the protein
specific polyclonal rabbit serum.
[0167] Construction of PAV-3 Recombinant Plasmids
[0168] The recombinant plasmid vectors were constructed by standard
procedures using restriction enzymes and other DNA modifying
enzymes.
[0169] i) Construction of Plasmid pFPAV211.
[0170] A 9.225 kb XhoI fragment (containing vector backbone plus
left [nt 004159] and right [nt 31053 to 34094] termini of PAV-3
genome) isolated from plasmid pFPAV200 (Reddy et al., 1999(a), J.
Gen. Virol. 80:563-570) was religated creating plasmid pPAVXhoIRL
(FIG. 9A). Nucleotide numbers of the PAV-3 genome referred to in
this report are according to GenBank accession no. AF083132 (and
are the same as in FIGS. 1-1 through 1-10). To delete the E1A
region, PAV-3 genome between nucleotides (nt) 0 to 531 was
amplified by using primers YZ-13 5'-ATA GGC GTA TCA CGA GGC-3' (SEQ
ID NO: 9) and YZ-14 5'-CTG GAC TAG TCT GTT CCG CTG AGA GAA AAC-3'
(SEQ IDS NO: 10), and plasmid pPAVXhoIRL DNA as a template in a PCR
reaction. The PAV-3 genomic DNA between nt 1231 and 1529 was
amplified by using primers YZ-15 5'-GTG GAC TAG TCTCAT GCA GCG
AACAAC C-3' (SEQ ID NO: 11) and YZ-16 5'-GTA CTA TCA CCT TCC TAA
GG-3' (SEQ ID NO: 12), and plasmid pPAVXhoIRL DNA as a template in
a PCR reaction. The product of first PCR was digested with
BamHI--SpeI and gel purified. The second PCR product was digested
with SpeI--Bsu36 and gel purified. The two gel purified fragments
were cloned into BamHI and Bsu36 digested plasmid pPAVXhoIRL in a
three-way ligation. The resulting plasmid pYZ20 carried 700 bp (nt
530 to 1230) deletion in E1A region and an engineered SpeI site.
The recombinant PAV-3 genome containing deletions in the E1A and E3
regions (pFPAV211) was generated by homologous DNA recombination in
E. coli BJ 5183 between XhoI linearized pYZ20 and genomic DNA of
PAV-3 E3 (Reddy et al., 1999(a), supra, FIG. 1B).
[0171] ii) Construction of Plasmid pFPAV212.
[0172] A 633 bp fragment (nt 827 to 1460) isolated by PCR
amplification (using oligonucleotides YZ-17 5'-ACA GTA ATG AGG AGG
ATA TC-3' (SEQ ID NO: 13) and YZ-18 5'-TAG GAC TAG TCC CAC AGA AAA
AGA AAA GG-3' (SEQ ID NO: 14) as primers and plasmid pPAVXhoIRL as
a template) was digested with EcoRV--SpeI and gel purified. A 403
bp fragment (nt 1820 to 2223 of PAV-3 genome) isolated by PCR
amplification (using oligonucleotides YZ-19 5'-ATG GAC TAG TCT TCT
GGT GCC GCC ACT A-3' (SEQ ID NO: 15) and YZ-20 5'-CCT AAT CTG CTC
AAA GCT G-3' (SEQ ID NO: 16) as primers and plasmid pPAVXhoIRL DNA
as a template) was digested with SpeI--Eco47III and gel purified. A
6.947 kb XhoI--StuI fragment of plasmid pPAVXhoIRL was blunt end
repaired with T4 polymerase and religated to create plasmid pYZ9a.
The two gel purified DNA fragments were ligated to EcoRV--Eco47III
digested plasmid pYZ9a in a three way ligation. The resulting
plasmid pYZ21 contains 360 bp deletion (nt 1460-1820) in
E1B.sup.small region and an engineered SpeI site. Finally, a 5.506
kb HpaI--AspI fragment of pYZ21 was ligated to 3.374 kb HpaI--AspI
fragment of pPAVXhoIRL to create plasmid pYZ21a. The recombinant
PAV-3 genome containing deletions in the E1B.sup.small and the E3
region (pFPAV212) was generated by homologous DNA recombination in
E. coli BJ5183 between XhoI linearized pYZ21a and the genomic DNA
from PAV E3 (Reddy et al., 1999(a), supra; FIG. 1C).
[0173] iii) Construction of Plasmid pFPAV507.
[0174] Plasmid pPAVXhoIRL was digested partially with Eco47III and
ligated to SpeI linker (triple phase stop [TPS] codon). Plasmid
pYZ9 containing SpeI linker inserted in E1B.sup.large ORF was
selected. The recombinant PAV-3 genome containing deletion in E3
and insertion in E1B.sup.large (pFPAV507) was generated by
homologous DNA recombination machinery in E. coli BJ5183 between
XhoI linearized pYZ9 and the genomic DNA from PAV E3 (Reddy et al.,
1999(a); FIG. 1D).
[0175] iv) Construction of Plasmid pFPAV214.
[0176] A 0.591 kb BamHI--AseI fragment was excised from plasmid
pYZ20 and ligated to 5.309 bp BamHI--AseI (partial) digested pYZ21
to create plasmid pYZ36. Finally, a 4.813 kb HpaI--AspI fragment
excised from plasmid pYZ36 was ligated to 3.373 kb HpaI--AspI
fragment of plasmid pPAVXhoIRL to create plasmid pYZ37. The
recombinant PAV-3 genome containing deletions in E1A, E1B.sup.small
and E3 region (pFPAV214) was generated by homologous recombination
in E. coli BJ5183 between XhoI linearized plasmid pYZ37 and genomic
DNA from PAV E3 (Reddy et al., 1999a; Fig. E). The full length
plasmid pFPAV214 contained 727 bp (nt 530-1230) deletion in E1A,
360 bp (nt 1460-1820) deletion in E1B.sup.small and 597 bp (nt
27405-28112) deletion in E3.
[0177] v) Construction of Plasmid pFPAV216.
[0178] Plasmid pYZ20 was digested with SpeI, blunt end repaired
with T4 polymerase and ligated to PmeI linker (GTTTAAAC) creating
plasmid pYZ39. A 1.424 kb AseI fragment of plasmid pYZ39 was
isolated and ligated to 6.774 kb AseI fragment of pYZ37 to create
plasmid pYZ40. Finally, a 1.730 kb NruI-PvuII fragment (containing
human cytomegalovirus (HCMV) immediate early promoter, GFP gene and
bovine growth hormone (BGH) poly(A) signal) was excised from
plasmid pYZ41a (Zhou et al., 2001, Virology) and ligated to PmeI
digested pYZ40 to create plasmid pYZ42. The recombinant PAV-3
genome containing GFP expression cassette insertion in E1A region
of E1A, E1B.sup.small and E3 deleted regions was generated by
homologous recombination in E. coli BJ5183 between XhoI linearized
pYZ42 and genomic DNA from PAV E3 (Reddy et al., 1999, supra)
[0179] Transfection and Isolation of PAV-3 Mutant Viruses
[0180] VIDO R1 cell monolayers seeded in 6-well plate were
transfected with 5 .mu.g of PacI-digested pFPAV211, pFPAV212,
pFPAV214, pFPAV216 or pFPAV507 recombinant plasmid DNAs using the
Lipofectin method (Gibco BRL). After 7-10 days of incubation at
37.degree. C., the transfected cells showing 50% cytopathic effects
were collected and freeze-thawed three times. Finally, the
recombinant virus was plaque purified and expanded in VIDO R1
cells.
[0181] Virus Growth Curve
[0182] VIDO R1 or ST cells were infected with mutant or wild-type
PAV-3 at an MOI of 5. The infected cells, harvested at indicated
times post infection were lysed in the infection medium by three
rounds of freeze-thaw. Virus titers were determined by serial
dilution infections of VIDO R1 cells followed by
immunohistochemical detection of DNA binding protein. Titers were
expressed as infectious unit (IU), in which 1 IU was defined as one
positive stained focus at 3 days post infection.
[0183] Western Blot
[0184] For Western blot, about 1.times.10.sup.6 VIDO R1 or Swine
Testicular (ST) cells (ATCC catalogue no. CRL 1746) were infected
with recombinant or wild-type PAV-3 at an MOI of 5. At indicated
times post infection, the cells were collected and lysed in 100
.mu.l of RIPA (0.15M NaCl, 50 mM Tris-HCl pH8.0, 1% NP-40, 1%
deoxycholate, 0.1% SDS). Proteins were resolved on SDS-PAGE under
the reducing condition and electrotransferred to nitrocellulose
membrane (Bio-Rad). Nonspecific binding sites were blocked with 1%
bovine serum albumin fraction V, and the membrane was probed with
the protein specific rabbit polyclonal serum. The membrane was
washed and exposed to goat anti-rabbit IgG conjugated to alkaline
phosphatase and developed using an alkaline phosphatase color
development kit (Bio-Rad).
[0185] Radioimmunoprecipitation
[0186] VIDO R1 cells in six well plates were infected with
wild-type PAV-3 at an MOI of 5. After virus adsorption for 1 h, the
cells were incubated in MEM containing 5% FBS. At different times
post-infection, the cells were incubated in methionine-cysteine
free MEM for 1 h before labeling with [.sup.35S]
methionine-cysteine (100 .mu.Ci/well). After 6 or 24 h of labeling,
the cells were harvested. Proteins were immunoprecipitated from
cells lysed with modified radioimmunoprecipitation (RIPA) buffer
and analyzed by SDS-PAGE as described previously (Tikoo et al.,
1993, J. Virol. 67:726-733).
[0187] Results
[0188] The results of the experimentation disclosed below indicate
that E1A is essential for virus replication and is required for the
activation of other PAV3 early genes; E1B.sup.small is not
essential for replication of PAV-3; and E1B.sup.large is essential
for virus replication. The results also demonstrate expression of a
desired transgene in a recombinant porcine adenovirus vector
comprising a deletion in E1A, E1B.sup.small and E3.
[0189] Characterization of PAV-3 E1 Proteins
[0190] In order to identify and characterize the proteins encoded
by E1 region of PAV-3, anti-E1A, anti-E1B.sup.small and
E1B.sup.large sera were produced by immunizing rabbits with 300 ug
of gel purified GST-protein (glutathione S-transferase) fusions.
Sera collected after the final boost was analysed by in vitro
transcription and translation assays to determine specificity of
the antibodies in the rabbit sera. The plasmids pSP64-PE1A,
pSP64-PE1Bs and pSP64-PE1B1 were generated in which coding sequence
of E1A, E1B.sup.small and E1B.sup.large respectively, was placed
downstream of the SP6 promoter (pSP64polyA vector containing SP6
promoter from Promega, Cat. No. P1241). In vitro translation of
pSP64-PE1A RNA resulted in the synthesis of a polypeptide of 35 kDa
(FIG. 10, lane 9), which was recognized by anti-E1A serum (FIG. 10,
lane 7). In vitro translation of pSP64-PE1Bs RNA resulted in the
synthesis of a polypeptide of 23 kDa (FIG. 10, lane 6) which was
recognized by anti-E1B.sup.small serum (FIG. 10, lane 4). Similarly
in vitro translation of pSP64-ElBl RNA resulted in the synthesis of
a polypeptide of 53 kDa (FIG. 10, lane 3), which was recognized by
anti-E1B.sup.large serum (FIG. 10, lane 1). These proteins were not
immunoprecipitated with anti-E1A serum (FIG. 10, lane 8),
anti-E1B.sup.small serum (FIG. 10, lane 5) or anti-E1B.sup.large
serum (FIG. 10, lane 2) from reactions in which pSP64polyA
(negative control plasmid) was translated in vitro.
[0191] To further characterize the proteins and to confirm the
specificity of the antisera, radioimmunoprecipitation assays were
performed. Anti-E1A serum detected a protein of 35 kDa in PAV-3
infected (FIG. 11A, lane 1-2) but not in mock-infected cells (FIG.
11A, lane 3). The 35 kDa protein was detected at 6 h (FIG. 11A,
lane 1) and 24 h (FIG. 11A, lane 2) post infection.
Anti-E1B.sup.small detected a protein of 23 kDa in PAV-3 infected
(FIG. 11B, lane 1-2) but not in mock infected (FIG. 11B, lane 3)
cells. The 23 kDa protein was detected at 6 h (FIG. 11B, lane 1)
and 24 h (FIG. 11B, lane 2) post infection. Similarly,
anti-E1B.sup.large serum detected a protein of 53 kDa in PAV-3
infected (FIG. 11C, lane 1-2) but not in mock infected cells. The
53 kDa protein was detected at 6 h (FIG. 11C, lane 1) and 24 h
(FIG. 11C, lane 2) post infection.
[0192] Generation of PAV-3 E1 Deletion/Insertional Mutants
[0193] Taking advantage of homologous recombination in E. coli
strain BJ5183, three full-length plasmids were constructed a)
pFPAV211 containing deletions in E1A (nt 530-1230) and E3 (nt
28112-28709) regions, b) pFPAV212 containing deletions in
E1B.sup.small (nt 1460-1820) and E3 (nt 28112-28709) regions and c)
pFPAV507 containing TPS codon in E1B.sup.large (nt 2190) and
deletion of E3 (nt 28112-28709) region (all nucleotide numbers are
with reference to FIG. 1). The PacI digested pFPAV211 or pFPAV212
plasmid DNAs were transfected into VIDO R1 cells and produced
cytopathic effects in 10-14 days. However, repeated transfection of
VIDO R1 cells with PacI digested pFPAV507 DNA did not produce any
cytopathic effects. The infected cell monolayers were collected and
freeze-thawed, and recombinant viruses were plaque purified and
propagated in VIDO R1 cells. The recombinant PAVs were named PAV211
(E1A+E3 deletion) and PAV212 (E1B.sup.small+E3 deletion). The viral
DNA was isolated from virus infected cells by Hirt extraction
method (Hirt, 1967, J. Mol. Biol. 26:365-369) and analysed by
agarose gel electrophoresis after digestion with restriction
enzymes. Since PAV211 and PAV212 genomes contain an additional SpeI
site in place of E1A or E1B.sup.small regions respectively, the
recombinant viral DNAs were digested with SpeI. As seen in FIG.
12A, compared with wild-type PAV-3 (lane 3), the PAV211 (lane 1) or
PAV212 (lane 2) genomes contain an additional expected band of 527
bp and 1463 bp respectively.
[0194] The ability of PAV211 and PAV212 to produce E1A and
E1B.sup.small or DNA binding protein (DBP) was tested by Western
blot analysis of these proteins from lysates of virus infected
Swine Testicular (ST) cells using PAV-3 E1A, E1B.sup.small or DBP
specific anti-serum. DBP anti-serum was prepared in the following
manner. A 900-bp fragment coding for the PAV-3 DBP (amino acids 102
to 457) was amplified by PCR using primers pDBP-3 (5'-CGG GAT CCG
GCC GCT GCT GCA GCT-3' (SEQ ID NO: 17)), pDBP-4 (5'-GCG TCG ACT CAA
AAC AGG CTC TCA T-3' (SEQ ID NO: 18)) and plasmid PAV3c63 (DBP
cDNA) (Reddy et al., 1998, Virology 251:414-426) DNA as a template.
The PCR fragment was digested with BamHI--SalI and ligated to
BamHI--SalI digested plasmid pGEX-5X-3 (Pharmacia Biotech) creating
plasmid pPDBPL8. This plasmid contains the coding region of DBP
(amino acids 102 to 457) fused in-frame to the C-terminus of
Schistosoma japonicum 26-kD glutathione S-transferase (GST)
gene.
[0195] Competent Escherichia coli BL21 were transformed with either
plasmid pPDBPL8 or plasmid pGEX-5X-3. Overnight cultures of 100 ml
LB broth were inoculated and grown until OD.sub.600 reached 0.5.
Cultures were induced for 4 h in 10 mM IPTG
(isopropyl-1-thio-.beta.-D-galactopyranoside). Cells were pelleted
and resuspended in 5 ml PBS. The cells were lysed by sonication and
the supernatant, collected after centrifugation was applied to GST
column. The matrix was washed by the addition of 10 bed volumes of
PBS and the fusion protein bound to the column was eluted in
glutathione elution buffer. The insoluble protein retained in the
cell pellet was purified by sodium dodecyl sulphate
(SDS)-polyacrylamide gel electrophoresis (PAGE). The area
containing the protein was excised and eluted by incubating the gel
slice in 20 ml water at 4.degree. C. overnight.
[0196] Rabbits were immunized subcutaneously with purified GST-DBP
fusion protein in freund's complete adjuvant followed by two
injections in Freund's incomplete adjuvant at two weeks interval
and DBP anti-serum was collected. Wild-type PAV-3 (FIG. 13C, lane
3) or PAV212 (FIG. 13C, lane 1) infected cells produced an E1A
protein of 35 kDa. No such protein was detected in PAV211 (FIG.
13C, lane 2) infected cells. Similarly, wild-type PAV-3 (FIG. 13B,
lane 3) and PAV212 (FIG. 13B, lane 1) produced a DBP protein of 50
kDa. No such protein was detected in PAV211 (FIG. 13B, lane 2)
infected cells. In addition, wild-type PAV-3 (FIG. 13A, lane 3)
infected cells produced an E1B.sup.small protein of 23 kDa (FIG.
13B, lane 3). However, no such protein was detected in PAV211 (FIG.
13A, lane 2) or PAV212 (FIG. 13A, lane 1) infected cells.
[0197] Construction of E1A+E1B.sup.small+E3 Deletion Mutant of
PAV-3
[0198] In order to increase insertion capacity of the PAV-3 vector,
a full length plasmid pFPAV214 carrying deletions in E1A (nt
530-1230), E1B.sup.small (nt 1460-1820) and E3 (nt 28112-28709) was
constructed by homologous recombination in E. coli BJ5183.
Transfection of VIDO R1 cells with PacI digested plasmid pFPAV214
DNA produced cytopathic effects in 7-10 days. The recombinant PAV-3
named PAV214 was plaque purified and expanded in VIDO R1 cells. The
viral DNA was extracted and analyzed by agarose gel electrophoresis
after digestion with NheI. As seen in FIG. 12B, the wild-type PAV-3
had a fragment of 1.430 kb (lane 2) that was missing in PAV214,
which instead had a fragment of 0.737 kb (lane 1).
[0199] Construction of E1A+E1B.sub.small+E3 deleted PAV-3
expressing GFP
[0200] In order to determine if PAV214 genome (E1A, E1B.sup.small
and E3 deleted) is useful for expression of foreign genes, a
recombinant PAV-3 expressing Green fluorescent protein (GFP) was
constructed. The full-length GFP gene (flanked by the HCMV promoter
and BGH poly (A) signal) was inserted into the E1A region of
pFPAV214 in the same transcriptional orientation as E1 (using the
homologous recombination machinery of E. coli) creating plasmid
pFPAV216. The PacI digested pFPAV216 DNA was transfected into VIDO
R1 cells to isolate recombinant virus PAV216. The viral DNA was
extracted and analysed by agarose gel electrophoresis after
digestion with restriction enzyme. Since there is an AseI site in
the CMV promoter, insertion of GFP transcription cassette in the
E1A region of PAV214 genome was confirmed by AseI digestion. As
seen in FIG. 12C, wild-type PAV-3 had a fragment of 1.274 kb (lane
1) that is missing in PAV216, which instead had two fragments of
0.584 kb and 1.739 kb (lane 2). Expression of GFP protein was
confirmed by Western blot using GFP specific polyclonal antibody
(Clonetech). As seen in FIG. 14, the GFP could be detected in
PAV216 infected VIDO R1 cells at 24 h.p.i. (lane 4) and 48 h.p.i.
(lane 5). The size of GFP expressed in cells infected with virus is
similar to that of purified GFP protein (lane 2), which is 28 kDa
in size. No such protein could be detected in mock-infected cells
(lane 1) or wild-type PAV-3 infected cells (lane 3).
[0201] Growth Kinetics of PAV211, PAV212, PAV214 and PAV216
[0202] In order to determine the importance of E1A and
E1B.sup.small in viral replication, the ability of mutant viruses
to grow in VIDO R1 cells and Swine Testicular (ST) cells was
compared to that of wild-type PAV-3. Virus infected cells were
harvested at different times point infection, freeze-thawed three
times and the cell lysates were analyzed for virus titer by DBP
detection assay. Virus titers were determined as infectious units
(IU) by qualitative DNA binding protein immuno-peroxidase staining.
The cell monolayers in 12-well plates were infected with serial
dilutions of virus. After adsorption of virus for 90 min, the cells
were washed and overlaid with MEM containing 2% FBS and 0.7%
agarose (Sigma, low melting temperature). On day 3 post infection,
the agarose overlay was carefully removed, the cells were
permeabilized with methanol/acetone (1:1 in volume) for 10 min at
-20.degree. C. and finally washed with PBS. Non-specific binding
sites were blocked by incubating the cells in PBS containing 1%
bovine serum albumin for 2 hr at room temperature. The blocking
solution was removed and rabbit anti-PAV-3 DBP serum diluted in PBS
was added to the plates. After 1 hr incubation at room temperature,
the plates were washed with PBS and then processed using Vectastain
Elite ABC kit (Vector Laboratories) containing biotinylated
anti-rabbit IgG and HRP-steptavidin complex. Finally, the reaction
was developed by the addition of substrate 3,3'-diaminobenzidine
(DAB) tetrahydrochloride. Titers were expressed as IU in which 1 IU
was defined as one positively stained cell/foci at 3 days post
infection. Virus titres were also determined using conventional
plaque assay.
[0203] Wild-type PAV-3 titer was 5.2.times.10.sup.7 IU\ml at 72 h
p.i. on VIDO R1 cells. The titers of mutant viruses were between
2.times.10.sup.7-3.2.times.10.sup.7 IU/ml, which are quite similar
to that of wild-type PAV-3 virus. Therefore, PAV vectors with
deletions in E1A and/or E1B.sup.small did not have any affect on
the ability of PAV-3 to propagate in VIDO R1 cells (E1
complementing cell line) (FIG. 15A). In contrast, we could not
observe any progeny virus production in PAV211, PAV214 and PAV216
infected ST cells (E1 non complementing). The virus titers at 72
h.p.i. were never more than 2.times.10.sup.5 IU/ml, which was lower
than the amount of input virus (FIG. 15B). All of these three
viruses carry deletions in E1A region. Most notably, mutant virus
PAV212 that carried deletions in E1B.sup.small region was able to
grow both in complementing and non-complementing cell lines (FIGS.
15A and 15B). At 72 h.p.i. the production of PAV212 in VIDO R1 and
ST cells were 3.3.times.10.sup.7 IU/ml and 3.9.times.10.sup.7 IU/ml
respectively.
Example 11
Generation of E1-Complementing Cell Line
[0204] The production of E1-deleted adenovirus vectors relies on
trans-complementation of the E1 functions in helper cells. Cell
line VIDO-R1 was generated by transformation of fetal porcine
retina cells with the plasmid DNA containing the E1 sequence of
HAdV-5 (Reddy et al., 1999; ATCC accession number PTA-155). Using
this complementing cell line the recombinant PAdV-3 with deletions
in E1A (nt 530-1230); E1B.sup.small (nt 1460-1820) and E3 nt
(28112-28709) has been rescued (Zhou and Tikoo, 2001, Virology,
291:68-76). However, attempts to rescue the recombinants with
increased deletion size were unsuccessful. We suggested that for
rescuing the E1-deleted PAdV-3 the E1B-large protein of PAdV-3 is
needed. To check this hypothesis, a new cell line, stably
expressing the gene for PAdV-3 E1B-large protein was developed.
[0205] The gene encoding PAdV 3 E1B large protein was cloned into
plREShyg vector. This vector contains the human CMV promoter, the
internal ribosome entry site (IRES) of the encephalomyocarditis
virus and hygromyrin B phosphotransferase gene. IRES permits the
translation of two open reading frames from one mRNA. VIDO-R1 cells
(fetal porcine retina cells transformed with HAdV 5 E1) were
transfected with pIREShygE1BL DNA and selected with hygromycin B.
About 20 days post-transfection hygromycin-resistant colonies were
observed. A. new cell line was established following single cell
cloning and designed VR1BL.
[0206] To study whether the cell line contains PAdV-3 E1B-large
sequence, integrated into the genome, Southern blotting analysis
was performed on total DNA extracted from the cells. As a probe,
the .sup.32P-labeled DNA of E1B-large gene was used. This probe
hybridized with the 1.9 kb--HindIII fragment of pIREShygE1BL,
containing the gene for PAdV-3 E1BL (large) (FIG. 16B) that has
been found in the genome of the VR1E1BL cell clones.
[0207] To study the PAdV-3 E1B-large gene expression in the VR1BL
cells, reverse transcriptase (RT) PCR was carried out using primers
specific to the portion of PAdV-3 E1B-large gene. From the RT-PCR,
a product of the expected size (317 bp) was obtained (FIG. 17). No
PCR product was observed in "no RT" control, suggesting that this
product came from mRNA template but not from DNA.
[0208] To confirm the expression of PAdV-3 E1BL protein, the VR1BL
cell line was subjected to immunofluorescence analysis, using
rabbit polyclonal antisera against PAdV-3 E1B-huge protein. The
VR1BL cells showed positive nuclear staining (FIGS. 18A-18B). At
the same time, parent VIDO-R1 cells were negative.
Example 12
Construction of the E1-Deleted Mutants of PAdV-3
[0209] Taking advantage of homologous recombination in E. coli
strain BJ5183, the plasmid pFPAV227 was constructed, containing
full-length genome of PAdV-3 with the deletion of E1 (nt 524-3274)
and a partial deletion of E3 (nt 28,112-28,709). Transfection of
VR1BL cells with PacI digested pFPAV227 DNA produced cytopathic
effect in 14 days.
[0210] Another plasmid called pFPAV219 contained the full-length
genome of PAdV-3 with the same deletions in the E1 and E3 regions,
but it had the insertion of 2320 bp DNA fragment, containing
GFP-expressing cassette (human CMV promoter, bovine growth hormone
poly(A) signal) in the E1 region. Transfection of VR1BL cells with
PacI digested pFPAV219 DNA also produced cytopathic effect in 14
days.
[0211] The recombinant viruses named PAV219 and PAV227 were
plaque-purified and expanded using VR1BL cell line. The viral DNA
was extracted from the infected cells and analyzed by agarose gel
electrophoresis after digestion with SpeI (FIG. 19). PAdV-3 has two
SpeI sites that give 724 by DNA fragment after digestion. PAV227
genome has an addition SpeI site that has been introduced in place
of E1 deletion. The SpeI-digestion of the PAV227 genome gives an
additional 527 by DNA fragment. The genome of PAV219 has two SpeI
sites in the GFP-expression cassette. The digestion with SpeI leads
to appearing the 849 bp and 547 bp DNA fragments.
[0212] To detect GFP expression by PAV219, ST cells were infected
with m.o.i. 1 TCID.sub.50/cell and 100 TCID.sub.50/cell. 24 h.p.i.
(hours post infection) the cells were harvested and FACS analysis
was performed. As seen in (FIG. 20), the infected cells were
GFP-positive and the expression was virus dose-dependent.
Example 13
Infection of Human Cell Lines with PAV219
[0213] To determine if human cell lines could successfully be
infected with recombinant PAdV-3 vector, the wide panel of
different human cell lines was infected with PAV219 at m.o.i. 100
TCID.sub.50/mo. 24 h.p.i. the cells were harvested and GFP
expressing cells were analyzed by FACS. The result of this
experiment is present in (FIG. 21).
[0214] Human embryo kidney 293 cell line is the best infectable
cell line. PAV219 infects 293 cell line as well as porcine ST cells
(an average 90% positive cells). PAV219 infects SAOS-2 osteosarcoma
well, too (68%). HeLa and Hep2 carcinomas, U118-MG glioblastoma and
MRC-5 lung fibroblasts could be infected with recombinant porcine
virus (from 47% to 26% positive cells in these cell lines). The low
infectable cell lines were A549 lung carcinoma and SK-N-MC
neuroblastoma.
[0215] Pre-existing neutralizing antibodies against adenoviruses in
the vast majority of the human population represent a major hurdle
to the use of human adenovirus derived vectors for gene delivery.
One of the ways to overcome this problem is a development of
non-human viral vectors for human vaccination and gene therapy. PAV
vectors disclosed herein can be used for human therapeutic and
prophylactic purposes. Antibodies against HAdV-5 do not neutralize
PAdV-3 in vitro and in vivo (Moffat et al., 2000, Virology,
272:159-167).
[0216] At present, adenovirus vectors are constructed by replacing
the essential E1 region with a foreign gene. It is necessary to
have E1 region deleted due to safety reasons. The proteins encoded
by this region interfere with the processes of cell division and
with the regulation of NF-.kappa.B and p53 (Russel, 2000, J. of
Gen. Virol. 81:2573-2604). The E1-deleted viruses are
replication-defective and therefore they must be propagated in a
cell line that expresses E1 proteins.
[0217] VIDO-R1 cell line (porcine retina cells, transformed with
HAdV-5 E1 (Reddy et al., 1999) can support the growth of
E1A+E1B-small deleted PAdV-3 (Zhou and Tikoo, 2001, supra). The
recombinant with insertional inactivation of the E1B-large could
not be rescued using VIDO-R1 (Zhou and Tikoo, 2001, supra). It is
possibly due to non-complementation of HAdV-5 55 kDa protein of the
PAdV-3 E1B-large defect.
[0218] VIDO-R1 cells were transformed with the plasmid containing
the gene for PAdV-3 E1B-large protein under control of human CMV
promoter. The gene was followed by IRES of the encephalomyocarditis
virus and hygromycin B phosphotransferase gene. This construct is
expected to be very effective for stable transfection because the
selective marker and gene of interest is translated from the same
mRNA. Indeed, all analyzed hygromicin-resistant clones were
positive for PAdV-3 E1B-large gene expression.
[0219] Using new VR1BL complementing cell line we rescued
recombinant PAV227. This virus lacks the E1 region (nt 524-3274)
and partially E3 (nt 28,112-28,709). This increases the safety of
the vector and increases the expected packaging capacity of PAdV-3
vector up to 5 kb of foreign DNA.
[0220] The construction of PAV219, a GFP-expressing recombinant,
further demonstrated the feasibility of using this vector system
for foreign gene expression. The construction of this recombinant
greatly facilitates the study of PAdV-3 infection of different
cultured cells and animals.
[0221] PAV219 was used to screen a panel of human cell lines for
the possibility of PAdV-3 infection. Human 293 cells were infected
as well as swine cells. SAOS-2 osteosarcoma cells were infected
very well with PAdV-3.
[0222] PAdV-3 did not infect A549 and Hep2 cells well that are well
infectable with HAdV-5 (Horwitz, 1996). For HAdV-5, virus
attachment to the cells is mediated by coxsackievirus and
adenovirus receptor (CAR) (Bergelson et al., 1997, Science
275:1320-1323; Tomko et al., 1997, P.N.A.S. USA, 94:3352-3356).
Without being bound by theory, the fact that PAdV-3 infects A459
and Hep2 cells poorly suggests that PAdV-3 uses a primary receptor
that is distinct from CAR. If PAdV-3 is using a receptor distinct
from CAR receptor, it is possible that some cells will be better
infected by PAdV-3 than HAdV-5 and vice versa. Some of the members
of Adenoviridae family use the primary receptor distinct from CAR
(Xu and Both, 1998, Virology, 248:156-163; Stevenson et al., 1995,
J. Virol. 69:2850-2857; Tan et al., 2001, J. Gen. Virol. 82:
1465-1472).
Example 14
Characterization of E4 Region
Materials and Methods
Cells and Viruses
[0223] The 6618 strain of PAV3 and all the mutant viruses were
cultivated in ST cell line. Eagle's Minimum Essential Medium (MEM)
with 2% fetal bovine serum (FBS) was used for growth of infected
cell. Virus stocks were prepared in ST cells and viral DNA were
extracted from the infected cells me the method of Hirt (1967). All
the virus stocks were prepared and tittered using ST cell line.
[0224] Construction of Recombinant Plasmid
[0225] The recombinant plasmid vectors were constructed by standard
procedures using restriction enzymes and other DNA-modifying
enzymes as directed by the manufacturers. In order to create
deletions in the PAV3 E4 region, plasmid pPAV200 containing the
full-length PAV3 genome in pPOLYSYN was digested by BamHI and the
5050 bp right terminal fragment was gel-purified and self-ligated
as plasmid pPAV400 which contains the whole E4 region of PAV3. A
set of deletion vectors which contain deletions of orfs in E4
region of PAV3 were constructed using plasmid pPAV400 and PCR
method. These deletion vectors were screened and determined using
different restriction enzymes. Later, these deletion vectors were
digested with restriction enzymes and the fragments with deletions
were gel-purified. Homologous recombination was carried out in BJ
5183 cell line using the deletion fragments and linearized
full-length genomic DNA. E4 modified full-length clones were
screened and determined by the digestion with different restriction
enzymes. The full-length clones with different deletions are shown
in FIG. 22.
[0226] Transfection of Cells
[0227] Monolayers of ST cells grown in 60 mm dish were transfected
with 5 or 7.5 ug of various PacI-digested recombinant full-length
plasmid DNA using Lipofectin (Gibco BRL). Following Transfection,
cells maintained in MEM containing 2% FBS at 37.degree. C. for
three to four weeks until cytopathic effects appeared. Cells
showing 80% CPE were harvested and freeze-thawed three times and
recombinant viruses were confirmed by restriction enzyme
analysis.
[0228] Polymerase Chain Reaction
[0229] PCR was carried out to verify the deletion created in the E4
mutant viruses. ST cells were infected with the various mutant
viruses and wild type PAV3, and viral DNA was extracted according
to the method of Hirt (1967). PCR products were generated by using
primers in the 5' and 3' flanking regions of the deletions. The 50
ul of PCR mix contained 0.2 pmol of each primer, 1.times. reaction
buffer, 0.2 mM dNTPs, 1 U pfu polymerase, and the viral DNA
template. The PCR procedure was designed with 35 cycles of
denaturation at 94.degree. C. for 30 seconds, annealing at
55.degree. C. for 30 s, and 72.degree. C. for 2 min. This was
preceded by an initial denaturing step of 94.degree. C. for 5 min
and completed by a final extension step of 72.degree. C. for 5 min.
The PCR products were analyzed by electrophoresis in a 1% agarose
gel and visualized with ethidium bromide. The results of PCR
analysis are shown in FIG. 24.
[0230] Virus Growth Curve
[0231] ST cells were infected with wild-type or mutant viruses at
10000 of TCID50 in six-well plate. The infected cells were
harvested at 12, 24, 36, 48 and 72 hours post infection, after
three rounds of freezing-thawing, virus lysis was titrated by
serial dilution infection of ST cells in 96-well plates and virus
titers were expressed at TCID.sub.50.
Example 15
Construction and Analysis of E4 Mutant Viruses
[0232] The E4 region encoded proteins of human adenoviruses show
redundant properties. For the purpose of analysis of porcine
adenovirus 3 E4 encoded proteins, a series of E4 mutant full-length
plasmids have been constructed. Initially, each of the E4 orfs were
deleted, separately, and then deletions of two neighbor orfs were
conducted. All the full-length mutant plasmids were cut using PacI
and the linearized plasmid DNAs were used for the transfection of
the ST cell line. A series of mutant viruses containing E4 orf1,
orf2, orf4, orf5, orf6, orf7, orf1&2, orf4&5, orf5&6,
orf6&7 were rescued from the transfected cells eight to fifteen
days later, however, we could not rescued viruses from the
transfection with the full-length plasmids containing the deletion
of orf3, orf2&3, orf3&4, even if we repeated the
transfection several times. The results of transfection in ST cells
are shown in Table 4.
TABLE-US-00007 TABLE 4 Results of the Transfections in ST Cells
Full-length plasmids Mutant viruses CPE pPAV200 PAV200 (WT) Yes
pPAV200d1 PAV401 Yes pPAV200d12 PAV412 Yes pPAV200d2 PAV402 Yes
pPAV200d23 PAV423 No pPAV200d3 PAV403 No pPAV200d34 PAV434 No
pPAV200d4 PAV404 Yes pPAV200d45 PAV445 Yes pPAV200d5 PAV405 Yes
pPAV200d56 PAV456 Yes pPAV200d6 PAV406 Yes pPAV200d67 PAV467 Yes
pPAV200d7 PAV407 Yes
[0233] The deletion size, location, inserted linkers, and the names
of the modified full-length plasmids and the mutant viruses are
summarized in Table 5. To determine the presence of the deletion in
the mutant viruses, both restriction enzyme digestion and PCR were
carried out. First, the viral DNAs were isolated from mutant virus
infected ST cells and digested with unique enzyme AvrII which is
the inserted linker. Two bands could be observed in the mutant
virus DNA samples and all the virus have the expected bands,
however, only one band could be seen in the wild-type PAV3 DNA
sample. The result of restriction enzyme analysis is shown in FIG.
23. Second, the specific deletions in the mutant viruses were
confirmed by PCR analysis. Three sets of PCR primers from the
flanking regions of the deletions were synthesized and mutant viral
DNA were PCR amplified and the PCR products were visualized on 1%
agarose gel. The shift of the size of PCR products from the mutant
viral DNA were observed compared to the wild-type PAV3 genomic DNA
and all of the mutant viral DNAs produced the expected smaller PCR
bands. The results of the PCR analyses are summarized in FIG.
24.
[0234] In vitro Analysis of PAV3 E4 Mutant Viruses
[0235] To analyze whether the single orf deletion or the combined
deletions had a noticeable effect on the capacity of PAV3 to
replicate in vitro, single step growth curve analysis of the mutant
viruses was conducted in ST cell line. ST cells were infected with
10.sup.4TCID.sup.50 of mutant viruses and the infected cells were
harvested at 12, 24, 36, 48 and 72 h post-infection. Virus lysate
from each sample was released by freeze-thawing three times and
titrated on ST cell line by analysis of the TCID.sup.50. Mutant
virus with deletion of orf 1, orf2, orf4, and orf1&2 grew
comparable efficiencies compared to wild-type PAV3. However, the
mutant viruses with deletion of orf 5, orf6, orf7, orf4&5,
orf5&6, orf6&7 grew a little slower compared to PAV3.
[0236] Table 5: Characterization of E4 Mutant Viruses. The table
summarizes the name of full-length plasmid with different
deletions, the open-reading frames deleted, the deletion region,
the deletion size, the linker inserted in the deletion region, the
name of the mutant viruses and the transfection results. CPE means
cytopathic effect.
TABLE-US-00008 TABLE 5 Characterization of E4 Mutant Viruses
Full-length Mutant Plasmidic Orfs Deleted Deletion Size Linker
Viruses CPE pPAV200d1 ORF1 (33436-33636) 201 AvrII PAV401 Yes
pPAV200d12 ORF1&2 (33044-33636) 593 AvrII PAV412 Yes pPAV200d2
ORF2 (33044-33404) 361 AvrII PAV402 Yes pPAV200d23 ORF2&3
(32737-33347) 611 SrfI PAV423 No pPAV200d3 ORF3 (32681-33036) 356
AvrII PAV403 No pPAV200d34 ORF3&4 (32264-33036) 773 AvrII
PAV434 No pPAV200d4 ORF4 (32264-32666) 403 AvrII PAV404 Yes
pPAV200d45 ORF4&5 (32103-32666) 564 AvrII PAV445 Yes pPAV200d5
ORF5 (32102-32248) 147 AvrII PAV405 Yes pPAV200d56 ORF5&6
(31834-32248) 415 AvrII PAV456 Yes pPAV200d6 ORF6 (31834-32053) 220
AvrII PAV406 Yes pPAV200d67 ORF6&7 (31303-32053) 751 AvrII
PAV467 Yes pPAV200d7 ORF7 (31303-31814) 512 AvrII PAV407 Yes
pPAV200 No No PAV200 Yes
[0237] Deposit of Biological Materials
[0238] The following materials were deposited with the ATCC:
Porcine embryonic retinal cells transformed with HAV-5 E1
sequences: VIDO R1 cells were deposited at the ATCC and have ATCC
accession number PTA 155.
[0239] The nucleotide sequences of the deposited materials are
incorporated by reference herein, as well as the sequences of the
polypeptides encoded thereby. In the event of any discrepancy
between a sequence expressly disclosed herein and a deposited
sequence, the deposited sequence is controlling.
[0240] While the foregoing invention has been described in some
detail by way of illustration and example for purposes of clarity
of understanding, it will be apparent to those skilled in the art
that various changes and modifications may be practiced without
departing from the spirit of the invention. Therefore the foregoing
descriptions and examples should not be construed as limiting the
scope of the invention.
Sequence CWU 1
1
18134094DNAPorcine Adenovirus Type 3 1catcatcaat aatataccgc
acacttttat tgcccctttt gtggcgtggt gattggcgga 60gagggttggg ggcggcgggc
ggtgattggt ggagaggggt gtgacgtagc gtgggaacgt 120gacgtcgcgt
gggaaaatga cgtgtgatga cgtcccgtgg gaacgggtca aagtccaagg
180ggaaggggtg gagccctggg gcggtcctcc gcggggcggg gccgagcggc
ggaaattccc 240gcacaggtgg agagtaccgc gggattttgt gccctctgga
ccggaccttc gccctccggt 300gtggcacttc cgcaccacac gtccgcggcc
cggtattccc cacctgacga cggtgacacc 360actcacctga gcggggtgtc
cttcgcgctg agaggtccgc ggcggccgcc cgagatgacg 420tgtgtgggtg
tattttttcc cctcagtgta tatagtccgc gcagcgcccg agagtcacta
480ctcttgagtc cgaagggagt agagttttct ctcagcggaa cagaccctcg
acatggcgaa 540cagacttcac ctggactggg acggaaaccc cgaggtggtg
ccggtgctgg aatgggaccc 600ggtggatctg cgcgacccct ctccggggga
tgagggcttc tgtgagccgt gctgggagag 660tctggtcgat ggactgccgg
acgagtggct ggacagtgtg gacgaggtgg aggtgattgt 720gactgagggg
ggtgagtcag aggacagtgg tgggagtgcc gctggtgact caggtggctc
780tcagggggtc tttgagatgg accccccaga agagggggac agtaatgagg
aggatatcag 840cgcggtggct gcggaggtgc tgtctgaact ggctgatgtg
gtgtttgagg acccacttgc 900gccaccctct ccgtttgtgt tggactgccc
cgaggtacct ggtgtgaact gccgctcttg 960tgattaccat cgctttcact
ccaaggaccc caatctgaag tgcagtctgt gctacatgag 1020gatgcatgcc
tttgctgtct atggtgagtg tttttggaca tttgtgggat tatgtggaaa
1080aaaaggaaaa agtgcttgta agaaatctca tgtgctattt cccatttttt
gtctttttag 1140aagctgtttc tccagcacct cacaggtcgg gttccccggg
acttggagac ctgccaggac 1200gcaagaggaa gtactgctat gactcatgca
gcgaacaacc tttggacctg tctatgaagc 1260gcccccgcga ttaatcatta
acctcaataa acagcatgtg atgatgactg attgtctgtg 1320tctctgccta
tatataccct tgtggtttgc agggaaggga tgtggtgact gagctattcc
1380tcagcatcat catcgctctg cttttttcta ctgcaggcta tttcttgcta
gctcgctgtc 1440ccttttcttt ttctgtgggc atggactatc aacttctggc
caagcttact aacgtgaact 1500accttaggaa ggtgatagta caggggtctc
agaactgccc ttggtggaaa aagatttttt 1560cggacaggtt tatcaaggta
gtagcagagg ccaggaggca gtacgggcaa gagttgattg 1620agatttttgt
ggagggtgag aggggctttg gtcctgagtt cctgcgggaa gggggactgt
1680acgaagaggc cgttctgaaa gagttggatt tcagcacctt gggacgcacc
gtagctagtg 1740tggctctggt ctgcttcatt tttgagaagc ttcagaagca
cagcgggtgg actgacgagg 1800gtattttaag tcttctggtg ccgccactat
gttccctgct ggaggcgcga atgatggcgg 1860agcaggtgcg gcaggggctg
tgcatcatca ggatgccgag cgcggagcgg gagatgctgt 1920tgcccagtgg
gtcatccggc agtggcagcg gggccgggat gcgggaccag gtggtgccca
1980agcgcccgcg ggagcaggaa gaggaggagg aggacgagga tgggatggaa
gcgagcgggc 2040gcaggctcga agggccggat ctggtttaga tcgccgccgg
cccgggggag cgggtggaga 2100ggggagcggg gaggaggcgg gggggtcttc
catggttagc tatcagcagg tgctttctga 2160gtatctggag agtcctctgg
agatgcatga gcgctacagc tttgagcaga ttaggcccta 2220tatgcttcag
ccgggggatg atctggggga gatgatagcc cagcacgcca aggtggagtt
2280gcagccgggc acggtgtacg agctgaggcg cccgatcacc atccgcagca
tgtgttacat 2340catcgggaac ggggccaaga tcaagattcg ggggaattac
acggagtaca tcaacataga 2400gccgcgtaac cacatgtgtt ccattgcggg
catgtggtcg gtgactatca cggatgtggt 2460ttttgatcgg gagctaccgg
cccggggtgg tctgatttta gccaacacgc acttcatcct 2520gcacggctgc
aacttcctgg gctttctggg ctcggtaata acggcgaacg ccgggggggt
2580ggtgcgggga tgctactttt tcgcctgcta caaggcgctg gaccaccggg
ggcggctgtg 2640gctgacggtg aacgagaaca cgtttgaaaa gtgtgtgtac
gcggtggtct ctgcggggcg 2700ttgcaggatc aagtacaact cctccctgtc
caccttctgc ttcttgcaca tgagctatac 2760gggcaagata gtggggaaca
gcatcatgag cccttacacg ttcagcgacg acccctacgt 2820ggacctggtg
tgctgccaga gcgggatggt gatgcccctg agcacggtgc acatcgctcc
2880ctcgtctcgc ctgccctacc ctgagttccg caagaatgtg ctcctccgca
gcaccatgtt 2940tgtgggcggc cgcctgggca gcttcagccc cagccgctgc
tcctacagct acagctccct 3000ggtggtggac gagcagtcct accggggtct
gagtgtgacc tgctgcttcg atcagacctg 3060tgagatgtac aagctgctgc
agtgtacgga ggcggacgag atggagacgg atacctctca 3120gcagtacgcc
tgcctgtgcg gggacaatca cccctggccg caggtgcggc agatgaaagt
3180gacagacgcg ctgcgggccc cccggtccct ggtgagctgc aactgggggg
agttcagcga 3240tgacgatgac tgaggatgag tcaccccctc ccctcctctt
gcaggtacgt ggccccgccc 3300agtgggatgg gctttggatg ggggaggggt
gttccctata aaagggggat gggggtggag 3360gcatgcagcc ccacggggaa
gcttgtgtgg aggatgtctt ccgagggtga gatccggacc 3420tgcttcattt
cagctcgtct tcccagctgg gccggcgtgc gtcagggagt ggccgggacg
3480aatgtgaacg gcggagtggt gggcgcccct gcccagagcg gggtgctggc
ctactcccgc 3540ttcgttcagc agcaacagca gcagccgggg acggcggcga
cggggtctgt gttccgggcg 3600gtgtttccat cggtggatct gagcgcggag
gtgggcatga tgcggcaggc gctggcggag 3660ctgcggcagc agctgcagga
gctgcgggag gtggtggaga tacagctgcg ggccacggcc 3720tcggaggcgg
ccgaggagga agaggaggag gagattgtgg tggacgagga ggtggcgccc
3780ggcgctggag cgaacaccat ggaagaggag gaggatgaga tggtcctgac
gatgactgtg 3840gtgggggacc ctgagcctgc tggagtggaa gcccagccgc
caccaccacc caccccggag 3900agcgaccctg cggtgcctgc tactaccact
accccgaagc ggctcagcta cggcgcgagc 3960aagaggagcg gtccatgcgc
ggaggacaac tgacgcggac tgtgggggga agaaggggga 4020ggaggaaaga
agaccatgga gacgggtgtt tgtctttttc cagcccaact ttattgagaa
4080taataataaa gcttatggat gtttggaacg ataatagcgt gtccagcgtt
ctctgtcttg 4140cagggtcttg tgtatcttct cgaggcaccg gtagacctgg
tgttggacgt tgaaatacat 4200gggcatgact ccctcggcgg ggtgcaggta
aagccactgg agggctgggt gcggggggca 4260ggtgcagtag atgatccagt
cataggcgtt ctggttgcgg tggtggttga aaatgtcctt 4320gaggagcagg
ctgatggcgg tgggcagacc cttggtgtag gcattgatga accggttgac
4380ctgggcgggc tgcatgaggg gggacatgat gtggtacttg gcctggatct
tgaggttgga 4440gatgttgccg ctctggtcgc ggcgggggtt catgttgtgg
aggacgacga ggacggcgta 4500gccggtgcag cgggggaagc gggcgtgcag
cttggagggg aaggcgtgga agaacttggc 4560gacccccttg tgtccgccga
ggtcctccat gcactcgtcg aggacgatgg cgatgggtcc 4620gcgggcggcg
gcgcgggcga agacgttgcg tgagtcagtg acatcatagt tgtgctcctg
4680catgaggtcc tggtagctca tgcggacaaa gtctggcatg agggtggcgg
tctgggggat 4740tagggtgtgg tccggaccgc tgcggtagtt gccctcgcag
atctgggtct cccaggcgac 4800tacctcctgc ggggggatca tgtccacctg
cggggtgatg aagaaaacag tctccggcgg 4860gggggagagg agttgggagg
agatgaggtt gcggagcagc tgggacttgc cggagccggt 4920gggaccgtag
atgacagcga tgactggctg gacctggtag ttgagggagc ggcaggtgcc
4980agccggggtg aggaagggca tgcaggcgtt gagggtgtcg cgcaggttgc
ggttctcttg 5040gacgaggtcc tgcaggaggt gtcggcctcc cagggagagg
aggtgggaga gggaggcgaa 5100ggccttgagg ggcttgaggc cctcggcgta
gggcatgtcc tgcagggcct ggtggagcac 5160gcgcatgcgc tcccagagct
cggttacatg tcccacggta tcgtcctcca gcaggtctgg 5220ttgtttctcg
ggttggggtt gctgcgtgag tacggaacga ggcggtgggc gtcgagcggg
5280tggagggtcc ggtccttcca gggccggagg gcccgcgtga gggtggtctc
ggtgacggtg 5340aagggggcgg tctggggctg ctcggtggcc agggtcctct
tgaggctgag gcggctggtg 5400ctgaaggtgg cgcttccgag ctgcgcgtcg
ttcaggtagc actggcggag gaggtcatag 5460gagaggtgtt gggtggcatg
gcccttggcg cggagcttgc cggggccgcg gtgcccgcaa 5520gcatcgcaaa
cggtgtcgcg cagggcgtag agcttggggg cgagcaggac cgtctcggag
5580ctgtgggcgt cgctgcggca gcgctcgcac tgggtctcgc actcgaccag
ccaggtgagc 5640tgggggttct ggggatcgaa gacgaggggg cccccgttcc
gcttgaggcg gtgtttacct 5700ttggtctcca tgagctcgcg tccggcgcgg
gtgaggaaga ggctgtcggt gtccccgtag 5760acggagcgca ggggccggtc
ggcgatgggg gtgccgcggt cgtcggcgta gaggatgagg 5820gcccactcgg
agatgaaggc acgcgcccag gcgaggacga agctggcgac ctgcgagggg
5880tagcggtcgt tgggcactaa tggcgaggcc tgctcgagcg tgtggagaca
gaggtcctcg 5940tcgtccgcgt ccaggaagtg gattggtcgc cagtggtagt
ccacgtgacc ggcttgcggg 6000tcggggggta taaaaggcgc gggccggggt
gcgtggccgt cagttgcttc gcaggcctcg 6060tcaccggagt ccgcgtctcc
ggcgtctcgc gctgcggctg catctgtggt cccggagtct 6120tcaggtgggt
acgctacgac aaagtccggg gtgacctcag cgctgaggtt gtctgtttct
6180atgaaggcgg aggagcggac ggagaggtcg ccgcgggcga tggcttcggt
ggtgcgggcg 6240tccatctggc tggcgaagac caccttctta ttgtcgaggc
gtgtggcgaa actgccgtag 6300agggcgttgg agagaagctt ggcgatgctg
cggagcgttt ggtttctgtc ccggtcggcc 6360ttttccttgg cagcgatgtt
gagctgcacg tagtctcggg cgaggcagcg ccactcgggg 6420aagatgctgt
tgcgctcgtc cggcaggagg cgcacggccc agccacggtt gtggagggtg
6480accacgtcca cggaggtggc tacctcgccg cggaggggct cgttggtcca
gcagaggcgg 6540ccgcccttgc gggagcagta ggggggcagg acgtccagct
ggtcctcgtc gggggggtcg 6600gcgtcgatgg tgaagagggc gggcaggagg
tcggggtcga agtagctgag gggctcgggg 6660ccgtcgaggc ggtcctgcca
gcggcgggcg gccagggcgc ggtcgaaggg gttgaggggt 6720tggccggcgg
ggaaggggtg ggtgagggcg ctggcataca tgccgcagat gtcatagacg
6780tagaggggct cccgcaggag gccgatgaag ttggggtagc agcggccgcc
gcgcaggctc 6840ttcgcggacg tagtcataca gctcgtggga gggcgcgagg
aggttcggcc gaggtgcggc 6900gcctggggcc ggctggcgcg gtagaggagc
tgcttgaaga tggcgtggga gttggagctg 6960atggtgggcc tctggaagac
attgaaggcg gcgtggggaa ggccggcctg cgtgtggacg 7020aaggcgcggt
aggactcttg cagcttgcgg accagacggg cggtgacgac gacgtcctgg
7080gcgcagtagc gcagggtggc ctggacgatg tcgtaagcgt ccccctggct
ctccttcttc 7140cacaggtcct tgttgaggag gtactcctga tcgctgtccc
agtacttggc gtgtgggaag 7200ccgtcctgat cgcgtaagta gtcccccgtg
cggtagaact cgttcacggc atcgtagggg 7260cagtgtccct tgtccacggc
cagctcgtag gccgcggcgg ccttgcggag gctggtgtgc 7320gtgagggcga
aggtgtcccg gaccatgaac ttgacgtact ggtgctgggg gtcctcgggg
7380gccatgacgc cctcctccca gtccgcgtag tcgcggcgcg ggcggaaggc
ggggttgggc 7440aggttgaagc tgatgtcatt gaagaggatg cggccgttgc
gcggcatgaa ggtgcgggtg 7500accaggaagg aggggggcac ctcgcggcgg
tgggcgagca cctgcgcggc caggacgatc 7560tcatcgaagc ccgagatgtt
gtggcccacg atgtagacct ccaggaagag gggcggcccg 7620cgcaggcggc
ggcgccgcag ctgggcatag gccagggggt cctcggggtc gtccggcagg
7680ccggggcccc gctcctgcgc cagctcggcg aggtctgggt tgtgggccag
caggtgctgc 7740cagagggtgt cggtgaggcg ggcctgcagg gcgtgccgca
gggccttgaa ggcgcggccg 7800atggcgcgct tctgcgggca gagcatgtag
aaggtgtggg ctcgggtctc cagcgctgca 7860ggcgggctct ggacggccac
cacctgcagc gcggcgtcca gcagctcctc gtcccccgag 7920aggtggaaga
ccagcaggaa gggcacgagc tgctttccga agcggccgtg ccaggtgtag
7980gtctccaggt cataggtgag gaagaggcgg cgggtgccct cgggggagcc
gatggggcgg 8040aaggcgatgg tctgccacca gtcggccgtc tggcgctgaa
cgtggtggaa gtagaagtcc 8100cggcggcgca cggagcaggt gtgggcggtc
tggaagatgc ggccgcagtg ctcgcacttc 8160tgggcctcct ggatgctctt
gatgaggtgg cagcggccct gggtgaagag caggcggagg 8220gggaagggga
ggcggggcgg cgggccctcg ggcggggggt cccagcgcac gtggtgcagg
8280tggtgttgct ggcgggtgac cacctggacg aaggtgggcc cggcggcgcg
ggccagctcc 8340accgcggtct ggggggtagc ctgcaggagg tcggggggcg
ggcgcaggag gtgcagctgg 8400aagaggttgg ccagggcgct gtcccagtgg
cggtggtagg tgatgctcca gctctccccg 8460tcctgggtgg tgccctggag
gcggagggtg gcgcggcgct cgagcaggag cccccgcgtg 8520ccggcctccg
cggcctcggc ggcggcggcc ggtctcaggc gggcagctgg gccaggggca
8580cgggcgcgtt gagctcgggc agcgggaggt ggtcgcggcg cagacgcgag
gcgtgggcga 8640tgacgcggcg gttgatgttc tggatctgcg ggttcccgga
gaagaccacg ggcccggtga 8700ctcggaacct gaaagagagt tccacggaat
caatgtcggc atcgtgggtg gccacctggc 8760gcaggatctc ggacacgtcc
ccgctgtttt cgcggtaggc gatgtcctgc atgaactgct 8820cgagctcgtc
ctcgtccagg tccccgtggc cggcgcgctc cacggtggcg gccaggtcga
8880cggtgatgcg gttcatgatg gccaccaggg cgttctctcc gttctcgttc
cacacgcgac 8940tgtagaccag ctggccgtcg gcgtcccgcg cgcgcatgac
tacctgggcc aggttgagcg 9000ccaccaggcg gttgaagggc gcctgcaggc
gcagggcgtg gtgcaggtag ttgagggtgg 9060tggcgatgtg ctcgcagagg
aagaagttta tgacccagcg gcgcagggtc agctcgttga 9120tgtcgcccag
gtcctcgagg cgctgcatga cccggtagaa ctcgggggcg aagcgaaaaa
9180actcgtgctg gcgggccgag accgtgagct cctcttccag ggcggcgatg
gcctcggcca 9240ccgcctgccg cacctcctcc tctaaggagg gcgggggcgt
gctgggtccg gccaccgccg 9300cctcttcttc ctcttctccc tccaggggtg
gcatctcctc gtcttcttct tctgctgctg 9360ctgcctccgc ggggacgggg
ggcgcaggcc ggggacggcg ccggcgcaag ggcagccggt 9420ccacgaagcg
ctcgatgacc tcgccccgca tgcggcgcat ggtctcggtg acggcgcggc
9480cgccctcccg gggccgcagc tcgaaggcgc ccccgcgcag cgcggtgccg
ctgcagaggg 9540gcaggctgag cgcactgatg atgcagcgtg tcaactctct
cgtaggtacc tcctgctgtt 9600gcagcgcttc ggcaaactcg cgcacctgct
cttcggaccc ggcgaagcgt tcgacgaagg 9660cgtctagcca gcaacagtcg
caaggtaagt tgagcgcggt gtgcgtcggg agccggaggt 9720gccggctgac
gaggaagtga aagtaggccg tcttgagctg ccggatggcg cgcaggaggg
9780tgaggtcttt gcggccggcg cgctgcaggc ggatgcggtc ggccatgccc
caggcctcct 9840gctggcagcg gccgatgtcc ttgagctgct cctgcagcag
atgtgccacg ggcacgtccc 9900ggtcggcgtc caggtgggtg cgaccgtagc
cccgcagggg gcgcagcagc gccaggtcgg 9960ccaccacgcg ctcggccagg
atggcctgct gcatgcgctg cagggagtct gagaagtcat 10020ccaggtccag
gaaccggtgg taggcgcccg tgttgatggt gtaggagcag ttgcccagca
10080cggaccagtt gaccacctgg tagtggggct ggatgacctc ggtgtagcgc
agtcgactgt 10140aggcgcgcgt gtcaaagatg taatcgttgc agaggcgcag
caggtgctgg tagcccacga 10200gcaggtgggg cggagggtag aggtagaggg
gccagtgttc cgtggccggt tggcgggggg 10260agaggttcat gagcatgagg
cggtggtagc ggtagatgaa gcgggacatc caggcgatgc 10320cgacggcgga
gacggaggcg cgggtccact ggtgggcgcg gttccaaatg ttgcgcaccg
10380ggcggaagag ctccacggtg taaatggatt gccccgtgag gcgggcgcag
tcgagggcgc 10440tctgtcaaaa agaaccgggt gtggttggtt ggtgtgtggt
agcgatctat ctttctttgt 10500gatcttggta gtgaagcctg ccaggctcca
gcagggggcg tccgccgtct ttccttcctt 10560ccctatctgg aggtgtgtct
ctgttctctt ttttatttca tgtagccatg catcccgttc 10620tgcggcagat
gaagccgccg gccggcgccc tgggcgcgga gggggcgacg cgctctcggt
10680cgccctcgcc gtcgctgacg cggccgcgcg aggaggggga gggcctggcg
cggctgtcgg 10740gcgcggcggc ccccgagcgg cacccacggg tgcagctcaa
gcgagaggcc atggaggcct 10800atgtgccgag gcagaatgcg ttccgcgagc
gaccggggga ggagggggag gagatgaggg 10860acctgcggtt ccgcgcgggg
cgggagatgc agctggaccg ggagcgagtg ctccagcccg 10920aggactttga
ggggcgcgtg gaggaggcgg ggggagtgag cgcggcgcgg gcccacatga
10980gcgcggccag cctggcccag gcctacgagc agacggtacg cgaggaggtc
aacttccaaa 11040agaccttcaa caacaacgtg cgcaccctgg tgagccggga
cgaggtgacc atgggactga 11100tgcacctgtg ggactttgtg gaggccttcc
tgcagcaccc ccggtcccgc gcgctgaccg 11160cgcagctgct gctgatcgcg
cagcactgcc gggacgaggg catggtgaag gaggcgctgc 11220tgagcctggg
cgcgcccgag agccgctggc tggtggacct ggtgaacctg ctccagacca
11280ttgtggtgca ggagcggtcc atgagcctga gcgagaaggt ggcggccatc
aactactcgg 11340tggcgaccct ggccaagcac tacgcgcgca agatctccac
cttctacatg cgcgcggtgg 11400tgaagctgct ggtgctggcc gacaacctgg
gcatgtaccg caacaagcgg ctggagcgcg 11460tggtcagcac ctcgcggcgg
cgcgagctca atgacaagga agctcatgtt tggcctccgc 11520cgggcgctgg
ccggggaggg cgaggaggac ctggaggagg aggaggacct ggaggaggcg
11580gaggaggagg agctggaaag aggaggagtt cggtccccgg ggaccgcggc
gcgtgaggtg 11640gcagtccccg ctgactgcga gcgatgaggg tgatgtgtac
tgatggcaac catccccctt 11700tttaacaaca acagcagcat ggcggcgagc
tctgaagctg gggcggcggc ggcgggggtg 11760agcgcggcct ccctggcgcc
cgagcgggcg acgcggatgc aggcgctgcc ctccctggac 11820gagccttggg
agcaggctct gcggcgcatc atggcgctga cggccgacgg gtctcggcgc
11880ttcgcgagcc agcccctggc caaccgcatc ggggccatcc tggaggcggt
ggtgcctccg 11940cgcacgaacc cgacgcacga gaaggtgctg accgtggtga
acgcgctgct ggagacctcg 12000gccatccgcc cggacgaggc cggcatggtg
tacgatgcgc tgctggagcg ggtctcccgc 12060tacaacagcg gcaacgtgca
gaccaacctg gaccggctgt cccaggacgt gcggcaggtg 12120atcgcccagc
gcgagcgctc gagcgccaac aacctgggca gcctggccgc gctgaatgcc
12180ttcatcgcct cgctgcccgc aacggtggag cggggccagg agagctacct
ggggttcctc 12240agcgcgctgc ggctgctggt gagcgaggtg ccgcagacgg
aggtgttccg ctcggggccg 12300cacaccttcc tgcaggcggc gcggaacggt
tccaagacgg tgaacctcaa ccaggccatg 12360gagaacctgc ggcccctgtg
ggggctgcag gcccccgctg gggagcgcgg gcacgtgtcc 12420tccctgctga
cgcccaacac ccggctgctg ctgctcctgg tggctccctt cgcggaggag
12480atgaacgtca gccggagctc ctacattggg cacctgctga cactctaccg
cgagacgctg 12540gccaacttgc atgtggacga gcgcacgtac caggagatca
ccagcgtcag ccgggcgttg 12600ggcgacgagg acgacgcggc gcggctgcag
gccaccctca acttcttcct gaccaaccgg 12660cagcggcggc tgccggcggc
gtatgccctg accgccgagg aggagcgcat cctgcgctac 12720gtgcagcagg
ccgtgagcct gtacctgatg caggacgggg cgacggccac gggcgccctg
12780gacgaggcca gccgcaacct ggagcccagc ttctacgcgg cgcaccggga
cttcatcaac 12840cgcctgatgg actacttcca tcgcgcggcc gcggtggcgc
ccaactactt tatgaatgcc 12900gtcctgaacc cccgctggct gccctcggag
ggcttcttca ccggcgtgta tgacttcccg 12960gagcaggacg agggggagga
gcggccctgg gacgcctttg acagcgacga ggagggccgc 13020ctcatgctgc
ggtccgcagc ctcctcagag ccctcctcct ccttcacccc cctgcccctg
13080accgaggagc cgccctcgcg gccctccacc ccggccctct cgcgcgtccc
gtcccgggca 13140tcctccctgc tctctctggc ctctctggga aagcgggagg
gaggggactc gctcgcctac 13200tcgccggcca cgcccaccta tggctctcgc
tggggctcgc gccgctccag cctggccagc 13260ggcgccgaca gcctggagtg
ggacgcgctg ctggcccctc ccaaggatgt gaacgagcac 13320ccaggcgccg
ccgccggccg ccgccgccgc gcctcccgct cctccctgga ggaggacatc
13380gacgccatca gcagccggct gttcacctgg cgcacgcgcg cccaggagat
gggcctgccc 13440gtggccagct tctcccgccg ccaccagccg cgccccgggg
ccctcgaaga cgacgaggag 13500gaggaagact ggcgccagga ccggttcttt
cgcttcgaag cgcccgagga aaaccccttc 13560cgccacatcg cccccaaggg
gctgtaatgc aaaaaagcaa aataaaaaac ccctcccggt 13620ccaactcacc
acggccatgg ttgtccttgt gtgcccgtca gatgaggagg atgatgccag
13680cagcgccgcc gcagggagcg tcgcctccgc cgtcctacga gagtgtggtg
gggtcttcgc 13740tcacggagcc tctttatgtg ccgccgcggt acctgggccc
caccgagggg cggaacagca 13800tccgttattc acagctcccg ccgctctacg
ataccacaaa gatctatctg atcgataaca 13860agtcggcgga tatcgccagt
ctgaactacc aaaacaacca cagtgacttt ctcaccagcg 13920tggtgcagaa
cagcgacttc acgcccatgg aggcgagcac gcagaccatc aacctggatg
13980agcgctcgcg ctggggcggg gagtttaaga gcattctgac caccaacatc
cccaacgtga 14040cccagtacat gttcagcaac agcttccggg tgcgcctgat
gagcgcgcgc gataaagaga 14100caaatgcccc cacctacgag tggttcaccc
tgaccctgcc cgagggcaac ttctcggaca 14160tcgcggtcat cgacctgatg
aacaacgcga tcgtggagaa ctacctggcg gtggggcggc 14220agcagggggt
caaggaggag gacatcgggg tgaagatcga cacgcgcaac ttccgcctgg
14280gctatgaccc ggagaccaag ctggtcatgc ccggcagcta caccaacatg
gcctttcacc 14340ccgacgtggt gctggcaccg ggctgcgcca tcgacttcac
cttctcccgc ctaaacaacc 14400tgctgggcat ccgcaagcgc tacccctacc
aggagggctt catgctgacc tacgaggacc 14460tggcgggggg caacatcccc
gcgctgctgg acctcaccac ctatgatcag gagaactcca 14520gcaccatcaa
gcccctgaag caggacagca agggtcgcag ctaccacgtg ggcgaggacc
14580ccgaggcggg ggacaccttc acctactacc gcagctggta cctggcctac
aactacgggg 14640acccggccac gggcaccgcc tcccagacgc tgctggtctc
cccggacgta acctgcggag 14700tggagcaggt ctactggagc ctgccggacc
tgatgcagga cccggtgacc ttccggccca 14760gccagacgcc gagcaactac
ccggtggtag ccacggagct actgccgctg cgctcccggg 14820ccttctacaa
cacccaggcc gtgtactccc agctcctgca gcaggccacc aacaacaccc
14880tggtctttaa ccgcttcccg gagaaccaga tcctcctgcg cccgccagag
tccaccatca 14940cctccatcag cgagaacgtg ccctcgctga cggaccacgg
cacgctgccg ctgcgtaaca 15000gcatccccgg ggtgcagcgg gtaaccgtca
ccgacgcgcg gcgccgcgtg tgtccctatg 15060tgtacaagag tctcggggtg
gtgaccccga gggtgctcag cagccgaacc ttctaaccga 15120cagccctacc
cgtcacaggg gagacagaga aaagacagcc agccccgcca tggccatcct
15180cgtctcgccc agcaacaact ttggctgggg actgggcctg cgctccatgt
acgggggcgc 15240ccgccgcctg tccccggatc accccgtgat cgtccgacgc
cactaccggg ccaactgggc 15300cagtctgaag ggacgcgtgg cccccagcac
catagcgaca acggatgacc ctgtggccga 15360cgtggtcaac gcgatcgccg
gcgccacccg ccgccggcgc cgccatcgtc gacgtcggag 15420ggccgcgcgc
gtctcctccg tggccgtcac cggggacccg gtggccgatg tggtcaacgc
15480ggtggaggcg gtagcccggc gccgccgcgc gcggcgccgt tcttcgcgca
tgcagaccac 15540gggggacccc gtggcggatg tggtggcggc ggtggaagcg
gtggcgcgcc ggaggcggag 15600cacccggcgg cggcgcaggc gctccgcgcc
ggccatcctg ggggtgcgcc gcagccgccg 15660cctccgcaaa cgcacctcgt
cctgagattt ttgtgttttg ttttttctgc ctcccgtggg 15720tgaacaagtc
catccatcca tccaacatcc gtggctgctg tgtctttgtc ttttctttgc
15780gttgcgcccc agttgagccg gcaccgacgc gctcggccat ggccatctcg
cgccgcgtga 15840aaaaggagct gctgcaggcg ttggcgcccg aggtgtacgg
ggcgcctaag aaggaggaga 15900aggacgtcaa agaggagtcc aaagctgacc
ttaaaccgct gaagaagcgg cgcaaggcca 15960agcgggggtt gagcgacagc
gacgaggtgc tggtgctggg cacgcgcccc aggcgccgct 16020ggacggggcg
gcgcgtgcgc gcccacctac cgcccggtgc cagcctcgcc tacgtcccgg
16080gtcttcggag gtcgagcgcc accaagcgct ctgcggacga gttgtatgcg
gacacggaca 16140tcctgcagca ggcgtcccag cgcctgaacg aatttgctta
tggcaagaga gcccggcggc 16200agcggcgggc ccgcccctcg ccgacccccg
cgtcccgcgg ccggaccacc aagcgctctt 16260atgacgaggt cgtggcagac
agtgacatcc tgcagcaact tggatccggg gaccgctcca 16320atgagttctc
ctatggcaag cggtcgctgc tgggggagtc aggagacacc gtcccggctg
16380tggccgtccc gctggaggaa ggcaggaacc acacacccag cctgcagccg
ctcaccgagc 16440ccatgcccct ggtgtcccct cgcacggccg tcaagcgccg
ggcgcccgcc gacgagccca 16500ccgcctcact ggtccccacc gtgcaggtcc
tggcccccaa gcgtcgtctg caggaggtgg 16560tggtggagcc gcccgctcca
gcacccacgc cgcccctagc cccgcggcgg tccagccggc 16620gcatcattct
ggctccgcgc cgggcgggcc ggccccaggc cgtcgtggcg ccgcagctca
16680gcgcggccgc ggcgctggag cgggcggcgg ccgccgtgcc cctgccaccg
gacacggagg 16740acgacctggt ggagatggca gaggctgtcg ccgcgcccga
ggtgctgccc agcctccccg 16800tctccatcat gccgcccacc gccacggagg
tggccctgcc cgtacagacc ccactgccgc 16860ccgtggcggt ggccaagagc
tccctgaccc ccggcctccg cgcgctgatg ggcaccgagc 16920gggtgccggt
tccagtcctg gaggcgcccc tggtggccat gcccgtgctc cgggccacca
16980ccgcccgtgc cgagcccccg cgccgcgtgc cccgcagggc cgtgcgggac
atcccggcca 17040ggcagccccg cacggtatcc ctgcccgtgc tcacggagcc
cggcccggcc accgcggtcg 17100cctccgtgcg cgcggcagcc caagtcctgc
aggcgccccc cgcccgaccg gccaccgtct 17160ccgtgggggt gggcaccgag
ccggtggtgc agtccatcac ggtcaagcgg tcaaagcgcc 17220tgaccaagca
ccatcggggt gcagaccatc gacgtcaccg tgcccaccgt ccgcactgtc
17280agcgtgggca ccaacacgcc ccggctgagg agcgcctcgg tgggcgtcca
gaccgctccc 17340gagacccgct cccagggggt gcaggtggct ttccaaccag
cgtgctagcc caccgcacac 17400ccaggcaggt gcggctgacg gcggtggtgc
cccccacccc gcgcgccccg gtggttccgg 17460tggcccggcg cccgcggcgg
ttccggtgcc tcccccagcc cctccagccc cgcgcgcgcc 17520gcgtgcgcct
cgcgccccca gagcgcctcg gcgtcgccgc cgtaccccgg tggcggtggc
17580agcgccgccc gcccgcagcg gcggtccccc gccctcggct gccgaggcgg
cccatcgtgc 17640tgcccggggt gcgctatcat cccagtcagg ccatggctcc
caccgcccaa cgcgtcatct 17700ggcgttgatt tatttttgga gacctgactg
tgttgtgttc cttaaatttt ttatcctcct 17760cctcctctgc tgaagccaga
cgatgctgac ctaccggttg cggctgcccg tgcggatgcg 17820gagaccgaga
ctccgcggtg ggttccgcgt ggcgcctcgg cgcagcggcg gcaggcggcg
17880gtaccgccgg gggccgatga ggggtggcat cctgccggcg ctggtgccca
tcatcgcggc 17940atccatctgg gccatccccg gcatcgcctc ggtggcgatg
agtgctagac aacgcaatta 18000acggcgctgc tgtgtatgtg tgtcttccat
gtgccttcct tccttcgttc ccaacggaac 18060agcagcaccg tctccatgga
ggacctaagc ttttccgcgt tggctccacg ctttggcacg 18120cggccggtca
tgggcacttg gagcgaaatc ggcacgagtc agatgaacgg cggcgcgctc
18180agctggagca atatctggag cgggctgaag agctttggta gttctctggc
ctccacggcc 18240aacaaggcct ggaacagcgg gacggtgacg agcgtgcgca
acaagttgaa ggatgccgac 18300gtgcagggga agataggtga ggtcattgcc
tccggggtcc acggtgccct ggacgtggcc 18360aaccaggccg tctcccacgc
cgtggaccgc cggtgcaaca gcagcagctg cggcagcagc 18420agctcctccg
ccagcagcag caacagatgg gcctcgtgga accctcctat gagatggaga
18480cagacgagct gcctcctccc cccgaggacc tcttgcctcc tcctcctcct
ccgccgcctg 18540cctcggccac tcccgcgcgc caatcccgcg ggacgtcccg
ccaagcgccc gccgccgccc 18600aggagatcat catccgctcc gacgagcccc
ctccctatga agagctgtat cccgacaagg 18660ccgggatccc cgccaccttg
gagctgcgtc ccgagaccaa actgcccgcc gtggcccaca 18720ataagatgcg
ccccccgccg ccgctcacca ccaccacctc ctccgctgcc gccgccgccc
18780ccgccccggc ccccgcggct cctgtgcgtc ggcgtccggc cgcggctccg
gccgcggctc 18840cggcgagttc caaaggcccc ccaggtgggg gtccgcgcgc
gcgggtggca aaacaaactc 18900aacaccattg tgggactggg tgtccgcaca
tgcaagcgcc gtcgttgtta ctgagagaga 18960cagcatggag aaacaacaat
gtctggattc aaataaagac acgcctattc ttccacggtg 19020ctccgcgctg
tgttattttc aacgggctgt ttccttttgc atctctgtgc catcgcgcca
19080cggggaattc cgcaggatgg cgacgccgtc gatgatgccg cagtggtcct
atatgcacat 19140ctccgggcag gacgcgtccg agtacctgtc tcccgggctg
gtgcagttct cccaggcgac 19200ggagacctac tttaacctga acaacaagtt
taggaacccc accgtcgcgc ccacccacga 19260tgtgacgacg gagcgctcgc
agcggctgca gctgcgcttc gtccccgtgg acaaggagga 19320cactcagtac
acatacaaga cccgcttcca gctggcggtg ggcgacaacc gcgtgttgga
19380catggcgagc accttctttg acatccgggg aacgctggac cggggaccct
ccttcaaacc 19440gtactcgggc accgcgtaca acatcatggc tcccaagagc
gctcccaaca actgtcaata 19500tctagaccct aaaggtgaaa ctgaggctgg
caaagttaat accattgctc aagcaagttt 19560tgtgggtcct attgatgaaa
ccacgggaga cattaaaatt acagaagaag aagacgaaga 19620gaccaccatc
gatcctttgt atgagcccca accccagctt ggtccaagct cgtggtcaga
19680caatatacct tctgcgacta gcggagctgg aagagttctc aaacagacca
caccgcgtca 19740accttgttac ggttcttatg cctctccgac aaatattcac
ggtgggcaaa cgaaggatga 19800caaggttaca ccattgtact ttacaaacaa
tcccgccacc gaagccgaag cactcgaaga 19860aaatggatta aagccaaatg
tcaccctata ctcagaggat gttgacctaa aagcaccaga 19920tactcatctg
gtctatgctg tgaatcaaac ccaggaattc gctcaatatg gacttggaca
19980acaggccgct ccaaacaggg ccaattacat cggcttcagg gacaacttta
tcgggctgtt 20040gtactacaac agcaatggca accagggcat gctagccggt
caggcctctc agctcaacgc 20100ggtggtcgac ctgcaggaca ggaatcaccg
aactagctac cagctcttcc tcgatagcct 20160ctatgacagg tcgaggtact
ttagcctgtg gaaccaggcc atcgattctt atgacaagga 20220tgtgcgtgtg
ctggaaaaca atggcgtgga ggacgagatg cccaactttt gctttcccat
20280cggcgccatc gagaccaaca tgacatttac acagctcaaa aagagtgaga
atggtggctc 20340aagagccaca acctggacaa aggagaatgg ggatgatggc
ggaaacggag cggagcacta 20400cctgggcatc ggcaacctca acgccatgga
gatcaatctc acggccaacc tctggcgcag 20460cttcctctac agcaacgtgg
cgctgtacct gcctgacaag tacaagtttt ccccgcccaa 20520cgtccccatc
gaccccaaca cgcactccta tgactacatc aacaagcgcc tgcccctcaa
20580caacctcatt gatacctttg tcaacatcgg ggcgcgctgg tccccggatg
tcatggacaa 20640cgtcaacccc ttcaaccacc accgcaacta cggcctgcgc
taccgctccc agctcctggg 20700caacggccgc tactgcaagt tccacatcca
ggtgccgcaa aagttctttg ccctcaagag 20760cctgctgctc ctgccggggg
cgacctacac ctacgagtgg tccttccgca aggacgtcaa 20820catgatcctc
cagtccacgc tgggcaacga cctccgcgcg gacggggcca aaatcaacat
20880cgagagcgtc aacctctacg ccagcttctt tcccatggcc cacaacaccg
cctccaccct 20940ggaggccatg ctgcgcaacg acaccaacaa ccaaaccttt
attgacttcc tctcctccgc 21000caacatgctc taccccatcc cggccaacgt
caccaacctg cccatctcca ttcccagccg 21060caactgggcc gccttccgcg
gctggagctt cacgcggctg aagcacaacg agacccccgc 21120cctgggctcg
cccttcgacc cctactttac ctactcgggc tccatcccct acctggacgg
21180gaccttctac ctgggccaca ccttccgccg catcagcatc cagttcgact
cctccgtggc 21240ctggccgggc aatgaccgcc tgctcactcc caacgagttc
gaggtcaagc gcaccgtgga 21300cggggagggc tacacggtgg cccagaccaa
catgaccaaa gactggttcc tggtgcagat 21360gctcgcccac tacaacatcg
gctaccaggg ataccacctg ccagagggct accgcgaccg 21420cacctactcc
ttcctgcgca actttgagcc catgtgccgc caggtgcccg actacgccaa
21480ccacaaagat gagtacctgg aggtgcccac caccaaccag ttcaacagca
gcggctttgt 21540atccgcggcc ttcaccgccg gcatgcgcga ggggcaccca
taccccgcca actggcccta 21600cccgctcatc ggcgaagacg ccgtgcagac
cgtgacccag cgcaagttcc tctgcgaccg 21660cacgctctgg cgcatcccct
tctcctccaa cttcatgtcc atgggcaccc tcaccgacct 21720gggccagaac
ctcctctacg ccaactcggc ccacgccctc gacatgacct tcgaggtcga
21780cgccatggat gaacccaccc tcttgtatgt tctgttcgag gtctttgacg
tctgcggcgt 21840gcaccagccg caccgaggcg tcatcgaggc cgtctacctg
cgcacgccct tctccgccgg 21900gaacgccacc acctaaggcg gagccgcgca
ggcatgggca gcaccgagga cgagctccga 21960gccatggcgc gcgacctcca
gctgccccgc ttcctgggca cctttgacaa gtccttcccg 22020ggcttcttgc
aagagtccca gcgctgctgc gccatcgtca acacggccgc ccgccacacc
22080ggaggccgcc actggctggc cgtcgcctgg gagcccgcct cgcgcacctt
ctacttcttt 22140gaccccttcg gcttctccga ccgggagctc gcccaggtct
atgactttga gtaccagcgc 22200ctgctgcgca agagcgccat ccagagcacc
ccggaccgct gcctcacgct cgtcaagagc 22260acccagagcg tgcagggacc
gcacagcgcc gcctgcggac tcttctgcct cctcttcctc 22320gccgcctttg
cccgctaccc cgacagcccc atggcctaca atcccgtcat ggacctggtg
22380gagggcgtgg acaacgagcg gctcttcgac gccgacgtcc agcccatctt
ccgcgccaac 22440caggaggcct gctacgcgtt cctcgctcgc cactccgcct
acttccgcgc ccaccgccac 22500gccatcatgg aacagacaca cctgcacaaa
gcgctcgata tgcaataaag gctttttatt 22560gtaagtcaaa aaggcctctt
ttatcctccg tcgcctgggg gtgtatgtag atggggggac 22620taggtgaacc
cggacccgcc gtcggctccc ctccatcccc tcttctctca aaacaggctc
22680tcatcgtcgt cctccgttcc cacggggaag atggtgttct gcacctggaa
ctggggcccc 22740cacttgaact cgggcaccgt cagtggaggc cgcgtctgca
tcagggcggc ccacatctgt 22800ttggtcagct gcagggccag catcacatcg
ggggcgctga tcttgaaatc acaattcttc 22860tgggggttgc cgcgcgaccc
gcggtacacc gggttgtagc actggaacac cagcaccgcg 22920gggtgggtca
cgctggccag aatcttgggg tcttccacca gctgggggtt cagcgccgcc
22980gacccgctca gcgcgaaggg ggtgatcttg caggtctgcc ggcccagcag
gggcacctgg 23040cggcagcccc agccgcagtc gcacaccagc ggcatcagca
ggtgcgtctc cgcgttgccc 23100atccgggggt agcaggcctt ctggaaagcc
ttgagctgct cgaaggcctg ctgcgccttg 23160gagccctccg agtagaagag
gccgcaggac cgcgccgaga aggtgttggg ggccgacccc 23220acgtcgtggc
tgcaacacat ggccccgtcg ttgcgcagct gcaccacgtt gcggccccag
23280cggttggtgg tgatcttggc gcgctcgggg gtctcgcgca gggcgcgctg
cccgttctcg 23340ctgttgagat ccatctccac cagctgctcc ttgttgatca
tgggcagccc gtgcaggcag 23400tgcagcccct ccgagccgct gcggtgctgc
cagatcacgc acccgcaggg gttccactcg 23460ggcgtcttca gacccgccgc
cttcaccaca aagtccagca ggaagcgggc catcactgtc 23520agcaggctct
tttgcgtgct gaaggtcagc tggcagctga tcttgcgctc gttcagccag
23580gcttgggccc cgcgccggaa gcactccagg gtgctgccgt ccggcagcag
cgtcaggccc 23640ttgacatcca ccttcagggg gaccagcatc tgcacagcca
gatccatggc ccgctgccac 23700ttctgctcct gagcatccag ctgcagcagc
ggccgggcca ccgccgggct cggggtcacc 23760gggcgcgggg ggcgggcccc
ctcctcttcc tccccatctt cgcccttcct cctcgcgggc 23820cgcgccgtcg
ccgctgccgt ctcttcagcc tcgtcctcct cctcctcgct gaccaggggc
23880ttggcacgcg cgcgcttccg ccgctcctgc acgggcggag aggccgcgcg
cttgcggcct 23940cccccgcgcc ggctgggggt cgcgacagga gcgtcgtcca
caatcagcac cccctcttcc 24000ccgctgtcat agtcagacac gtccgaatag
cggcgactca ttttgcttcc cctagatgga 24060agaccagcac agcgcagcca
gtgagctggg gtcctccgcg gccccgaccc ttccgccgcc 24120accaccgccg
ccacctccgc ccacgtcacc gccaccttca ctgcagcagc ggcagcagga
24180gcccaccgaa accgatgacg cggaggacac ctgctcctcg tcctcctcgt
cctccgcctc 24240cagcgagtgc ttcgtctcgc cgctggaaga cacgagctcc
gaggactcgg cggacacggt 24300gctcccctcc gagccccgcc gggacgagga
ggagcaggag gaggactcgc ccgaccgcta 24360catggacgcg gacgtgctgc
agcgccacct gctgcgccag agtaccatcc tgcgccaggt 24420cctgcaggag
gccgcccccg gcgcagccgc ggaggccgcc gaggcgccct cggtggcgga
24480gctcagccgc cgcctggaag cggccctctt ctcccccgcc acgccgccgc
ggcgccagga 24540gaacggaacc tgcgccccgg acccccgcct caacttctac
ccggtcttca tgctgcccga 24600ggccctggcc acctacctcc tcttcttcca
caaccaaaag atccccgtca gctgccgcgc 24660caaccgccca cgagccgacg
cgcactggcg gctgcccagt gggaccccct tacctgacta 24720tccaaccacc
gacgaggttt acaagatctt tgagggcctg ggggacgagg agccggcctg
24780cgccaaccag gacctgaaag agcgcgacag cgtgttagtc gagctcaagc
tggacaaccc 24840ccgcctggcg gtggtcaagc agtgcatcgc cgtcacccac
ttcgcctacc cggccctggc 24900gctgccaccc aaggtcatga gcacgctcat
gcagaccctg ctggtgcgcc gcgcgagccc 24960actccccgac gagggcgaga
cgcccctcga ggacctcctg gtggtcagcg acgagcagct 25020ggcccgctgg
atgcacacct cggaccccaa ggtcctggag gagcggcgca agaccgtcac
25080cgccgcctgc atggtcacgg tgcagctcca ctgcatgcac accttcctca
cctcccgcga 25140gatggtgcgc cgcctcggag agtgcctcca ctacatgttc
cgccagggct acgtcaagct 25200agctagcaag atcgccaata tggaactctc
taacctggtc tcctacttgg gcatgctgca 25260cgaaaacagg ctcggtcagc
acgtgctcca ccacaccctc aagcatgagg cgagacgcga 25320ctacgtccgg
gacaccattt acctatacct ggtctatacc tggcagaccg ccatgggggt
25380ctggcagcag tgcctcgagg accgaaacct gcgcgccctg gaaacgtctc
tggctcgcgc 25440tcgccagagc ctgtggacgg gctttgatga gcgcactatc
gcgcaggacc tcgccgcgtt 25500ccttttcccc accaagctcg tagagaccct
gcagcgctcg ctccccgact ttgccagcca 25560gagcatgatg catgccttcc
gctccttcgt cctcgagcgc tccggcatcc tgcccgccgt 25620ctgcaacgcg
ctcccctctg actttgtgcc caccgtctac cgcgagtgcc cgccgcccct
25680ctgggctcac tgctacctcc tgcgcctcgc caacttcctc atgtaccact
gcgacctcgc 25740cgaggacacc tccggcgagg gcctctttga gtgctactgc
cgctgcaacc tctgcgcacc 25800gcaccgctgc ctcgccacca acaccgccct
cctcaacgag gtgcaagcca tcaacacctt 25860tgagctccag cggcccccca
agcccgacgg caccctgcca ccgcccttca agctgacccc 25920cggtctctgg
acctccgcct tcctccgcca ctttgtctcc gaggactacc actcggaccg
25980catcctcttc tacgaggacg tgtcccgccc ccccagggtg gagccctccg
cctgcgtcat 26040cacgcactcg gccattctcg cgcaattgca tgacatcaaa
aaggccaggg aagagttttt 26100gctgaccaaa ggccacggcg tctacctaga
cccccacacc ggagaggagc tcaacaccgc 26160cgccccgtcc accgcccacc
atgccgcccc tccggaggaa gcccatccgc agcagcacca 26220gcaccagcag
cagccgagcc accgccgccg ccaccaccgc tccagctacg cagaccgtgt
26280ccgaagcgag ctccacgcct acggcggtgc gaccggttcc tcccgcgacc
ctgtctctgg 26340cggatgctct gccagaggaa cccactcccg cgatgctgct
cgaagaagag gctctcagca 26400gcgagaccag cggcagctcc gaaggcagtt
tgctcagtac cctcgaggaa ctggaggagg 26460aggaggaacc ggtcacaccg
acgaggccat ccaagccctc ctacaccaac agcagcagca 26520gcaagagcat
cagccagcgc aggaactccg tcgtccccag cgaggctcgt agatggaatc
26580agacatccat ccaccggagt agccagccag gtaggacacc tccgccctcg
gcccgccgac 26640gctcctggcg ccgctaccgc cacgacatcc tctcggccct
ggagtactgc gccggagacg 26700gagcctgcgt gcgccggtac ctactctacc
accacaacat caacatccct tccaagatca 26760tccgttacta caaatcctct
tcccgttcca gcgatctcca ggaaggccgc agcagcggcg 26820gcagcagaac
cagcccacgt cagccagctg agagctaaga tcttccccac gctgtacgcc
26880atcttccagc agagccgcgg cggccaggac gccctcaaaa tcaggaaccg
caccctgcgc 26940tccctcacca agagctgtct gtatcaccgc gaggaggcca
agctggaacg cacgctctcg 27000gacgcagaag ctctcttcga gaagtactgc
gctcggcagc ggcagacccg ccggtattta 27060aggagcggac cctgcgtgcg
gacacaccat gagcaaacaa atccccaccc cgtacatgtg 27120gtcttatcag
ccacaatctg ggcgtgccgc cggtgcctcc gtcgattact ccacccgcat
27180gaattggctc agtgccgggc cttccatgat tggccaggtc aatgacatcc
gacacaccag 27240gaaccagatt ctcattcgcc aggcccttat caccgagacg
ccacgccccg tccaaaatcc 27300cccgtcctgg cccgccagcc tgttgcctca
gatgacgcaa ccgcccaccc acctgcacct 27360gccgcgtaac gaaattttgg
aaggcagact gactgacgcc ggcatgcaat tagccggggg 27420cggagccctc
gcacccagag acttatatgc cctgaccctc cgcggcagag gcatccagct
27480caacgaggac ctacccctct cggcgagcac tctccggccg gacggcatct
tccagctcgg 27540aggcggaggc cgctcctcct tcaaccccac cgacgcctac
ctgacgctgc agaactccag 27600ctcccttccc cgcagcggcg gcatcggcag
cgagcaattt gtccgcgagt tcgtgcccac 27660ggtctacatc aaccccttct
ccggaccgcc cgggacctac cccgaccagt tcatcgccaa 27720ctacaacatc
ctaacggact ctgtagcagg ctatgactga cggtccccag ggtcagcagc
27780ggctgcggga gctcctcgac cagcaccgcc gccagtgccc taaccgctgc
tgcttcgcca 27840gggaagggat tcacccggag tacttttgca tcacccgcga
gcactttgag gccgagtgca 27900tccccgactc tctgcaagaa ggccacggtc
tgcgcttcag cctccccacg cgctacagcg 27960accgccgcca ccgcgatgga
gaccgcacca tcctcacttc gtactactgc ggccctgctt 28020ctttcaaagt
tcgctgtctc tgcggccatc ctgctcctca ccctcttctt ctcgaccttc
28080tgtgtgagct gtacaaccgc tcgtagcgtc agcccctaca cctcccctcg
cgtccaattt 28140ctgtccgaca tagaaccaga ctctgactct tactcgggct
ctggctctgg ggacgatgaa 28200gattatgaat atgagctggc taccaacaca
ccgaacgaag acattctagg cagcatagtc 28260atcaacaacc agatcgggcc
caagaccctg gccctgggat acttttatgc cgccatgcag 28320tttgtcttct
ttgccatcat catcatcgtc ctcatcctct actaccgccg ctacgtgctg
28380gccaccgccc tcatcgtgca gcgccagatg tggtcctccg aggccgtcct
gcggaaaacc 28440ttctcggcca ccgttgtggt tactccccca aaacaagtca
ccccctgcaa ctgctcctgc 28500cgcttcgagg agatggtgtt ctactacacc
acctccgtct tcatgccctg gtgggcctca 28560tcctcctgct caccgccatg
gtccgcctgg ccaactggat agtggatcag atgcccagca 28620ggaaccgcgc
cccgccgctg ccaccgcccc tcacctatgt gggaccctgc gccgaggacc
28680acatctacga tgagccaacc gtagggcaat acgtacagat gaagtagctc
cccctctttc 28740ccattccccc atttttctct attcaataaa gttgcttacc
tgagttcatc cacactcggt 28800ctgccagtgc agtctatcca tgcgccgttt
tccatactca catagcgcag ccgcgcacgc 28860ctcgccaggt gacgaaactg
tcgaaatgta acatttcgcg cttctgtcag cagcaccccg 28920ttatagacca
gttccaccat gggaccgaag aagcagaagc gcgagctacc cgaggacttc
28980gatccagtct acccctatga cgtcccgcag ctgcagatca atccaccctt
cgtcagcggg 29040gacggattca accaatccgt ggacggggtg ctgtccctgc
acatcgcacc gcccctcgtt 29100tttgacaaca ccagggccct caccctggcc
ttcgggggag gtctacagct ctcgggcaag 29160cagctcgtcg ttgccaccga
gggctcgggg ctaaccacca acccggatgg caagctggtt 29220ctcaaagtca
agtcccccat caccctgacc gccgagggca tctccctgtc cctgggtccc
29280ggtctttcta actcagagac cggcctcagt ctgcaagtca cagctcccct
gcagttccag 29340ggcaacgccc tcactcttcc cctcgccgcc ggtctccaaa
acaccgatgg tggaatgggt 29400gtcaaactgg ggagcggtct caccacggac
aacagtcagg cggtgaccgt tcaggtggga 29460aatggacttc agctgaacgg
cgaaggacaa ctcaccgtcc ccgccacggc ccctttagtc 29520tcagggagcg
caggcatctc tttcaactac tccagcaatg acttcgtctt agacaatgac
29580agtctcagtt tgaggccaaa ggccatctct gtcacccctc cgctgcagtc
cacagaggac 29640acaatctccc tgaattattc taacgacttt tctgtggaca
atggcgccct caccttggct 29700ccaactttca aaccctacac gctgtggact
ggcgcctcac ccacagcaaa tgtcattcta 29760acaaacacca ccactcccaa
cggcaccttt ttcctatgcc tgacacgtgt gggtgggtta 29820gttttgggtt
cctttgccct gaaatcatcc atcgacctta ctagtatgac caaaaaggtc
29880aattttattt ttgatggggc aggtcggctt cagtcagact ccacttataa
agggagattt 29940ggatttagat ccaacgacag cgtaattgaa cccacagccg
caggactcag tccagcctgg 30000ttaatgccaa gcacctttat ttatccacgc
aacacctccg gttcttccct aacatcattt 30060gtatacatta atcagacata
tgtgcatgtg
gacatcaagg taaacacact ctctacaaac 30120ggatatagcc tagaatttaa
ctttcaaaac atgagcttct ccgccccctt ctccacctcc 30180tacgggacct
tctgctacgt gccccgaagg acaactcacc gtccccgcca cggccccttt
30240agtctcaggg agcgcaggca tctctttcaa ctactccagc aatgacttcg
tcttagacaa 30300tgacagtctc agtttgaggc caaaggccat ctctgtcacc
cctccgctgc agtccacaga 30360ggacacaatc tccctgaatt attctaacga
cttttctgtg gacaatggcg ccctcacctt 30420ggctccaact ttcaaaccct
acacgctgtg gactggcgcc tcacccacag caaatgtcat 30480tctaacaaac
accaccactc ccaacggcac ctttttccta tgcctgacac gtgtgggtgg
30540gttagttttg ggttcctttg ccctgaaatc atccatcgac cttactagta
tgaccaaaaa 30600ggtcaatttt atttttgatg gggcaggtcg gcttcagtca
gactccactt ataaagggag 30660atttggattt agatccaacg acagcgtaat
tgaacccaca gccgcaggac tcagtccagc 30720ctggttaatg ccaagcacct
ttatttatcc acgcaacacc tccggttctt ccctaacatc 30780atttgtatac
attaatcaga catatgtgca tgtggacatc aaggtaaaca cactctctac
30840aaacggatat agcctagaat ttaactttca aaacatgagc ttctccgccc
ccttctccac 30900ctcctacggg accttctgct acgtgcccca gagtgcctag
agaaccctgg ccgtcagccg 30960gcctccccct tcccaggcca cccggtacac
cacccgctcc atgtttctgt atgtgttctc 31020ctcccgccgc ttgtgcagca
ccacctcccg ctgctcgagc tgaggatccg tgatggacac 31080aaagccagga
agacacatcc tcagctccgt gggggcgtcc aacaactgtt tatgtaaagg
31140aaaataaaga ctcagagaaa atccaagttc atatgatttt tcttttattg
attgggggaa 31200ttgattcagg tggggtgtgc ataatcacaa aaatcacatc
agcaggtaca cacctgagac 31260atcagacagg ggtaaggaca gcgcctcagc
ttctggaaca gacatcagaa atatttaatc 31320tgctggtagc taacactcct
tcccaacacc atacactcct ggagggccct ctgcctctcc 31380tcctcccgct
ccgcgtccct ctgccgggac caccactccc cctccgtgaa ctgctgcttc
31440ctcccccgcc gctgcgcccc gatggcctcc gccgccagct tcagccagtg
ccgcaagcgc 31500tgggcgcagc gccgagccac cggctcgctc agctcgtggc
agcgccggca caccagcact 31560atgtaattgg catagtcccc gtcacagtag
atgacctccc cccagtggaa catgcgcaac 31620agcttcagat cacagtcata
catgatcttt atgtacatca ggtgggcgcc tcgaaacatc 31680acactgccca
cgtacatcac gcgactcacg ctgggcaggt tcaccgcctc cctgaaccac
31740cagaagatgc gattgtactc gcagccccgg atgatctcgc gcatcaggga
gcgcatcacc 31800acctgccccg cgcggcactc cagactggac cttttcagac
agtggcaatg aaagttccac 31860agcgtcgcgc ccgcacagcg tctccgggct
gaaacatatc tgctccagct ccaacccccc 31920acacaggctg tactgcagga
aaatccattc ttgatgggaa aggatgtagc gccaggggac 31980cacaatctcc
aaacagggaa caaaacatac cgcggcccgg ctgttgcgca cggcccccac
32040cggatgcaac gtgctcacgg agcagatacg ggtgggacag cggcccacgt
ctcatagcaa 32100gtcaagtccg gaagtggcac ggggttcgcc accactgcta
ctgctgccgc tgcgccacca 32160gctccatcgg ctcctccatc ctcctcctgt
tccatcggct gaggtgctgc ctcctcctcc 32220tcctgccgct gctccatcat
gctcgtctgc ggtcatcagg agtcaaaaaa ttcattggcc 32280accgcacgca
gagagaacat ggagcgcagg ggcccaggtg cccggcccgt gcgctcgctc
32340aactccccca gcaggtactc atagagatgc tcctccaaat ccaccgcaaa
ccaggcatgc 32400agaaactctt ccgttcgagg accgcccacg gtaaagacat
agccctcccg caccttcacc 32460gctgccagct gcacgcgctc atgtcgctgg
gagtacaccc ggacccgggc ctggatgtac 32520tccagcacct gatcgctcag
acacctcaca gagatgccag cctgagccag cttctcatag 32580agaggtggct
gaatcttgag cttgaagcag cgagcggcta ggcactcccc gcccccttgg
32640aacagggcgg ccgggtcagc catggacttc ctctacatcc ggggtcctgg
ccacctcaca 32700aactatctgg ccaatcgcct gaccacgggt caccaggtaa
ggatgatgtc cgttgttgcg 32760aatgagaatg ctcagaggtg actcggtagc
gttatcaatc acgtccccaa aggtccaaag 32820gtcccagtta gaagtcaggt
gcttcagacc gcagacacgc ccatagcaac cagtgggaaa 32880agccagcaag
agatccgtgg gcacatgcac cgaagctccc gcaggaatct ccacccactc
32940cgaggcgtag accgtgtaag ctacacaccc cgcctcccga gtgggagcag
aagcattctc 33000gctcagccga aagaacttca gggtggcctg catatcctct
tttactcact tgttagcagc 33060tccacacaga ccagggttgt gttggcggga
ataggcagca ggggtacgtc cccagtgagg 33120gacacctgga tggggggcag
aggattgatg ccaggaagca gcaggtactg ggaaacagag 33180accagatccc
tcctctgaaa aatctcgctc agtcggacaa acacagcaaa cccagtgggc
33240acgtagacta gcacattaaa aaggatcacg ctgggctgtt ctgacgtcag
caccagatgt 33300cgggacgtgc gcagatgaat gcggttctga tgaattaccg
gaggcctctc acccgcagcc 33360aacagcagac cgggctgctg atgcggtccc
gcagacatat atgagttcaa tgtgtgtctt 33420ttttctaaac gtctagtgag
tgtgctcgtc ctgctcctgc caatcaaaat ccgggcacca 33480gggctggtgg
ttggacccga tgaagaagcg aggagaggcg gcctcctgag tgtgaagagt
33540gtcccgatcc tgccacgcga ggtaggcgaa gtacagatag agcacggcga
gaacagtcag 33600caccgcggcc agcagcagtc ggtcgtgggc catgagaggg
ggctgatggg aagatggccg 33660gtgactcctc tcgccccgct ttcggtttct
cctcgtctcg ctctcagtgt ctctctctgt 33720gtcagcgccg agacgagtgt
gagcgaacac cgcgagcggg ccggtgatat acccacagcg 33780gatgtggcca
cgcctgcggt cggttaatca gtaccccatc gtccgatcgg aattcccccg
33840cctccgcgtt aacgattaac ccgcccagaa gtcccgggaa ttcccgccag
ccggctccgc 33900cgcgacctgc gactttgacc ccgcccctcg gactttgacc
gttcccacgc cacgtcattt 33960tcccacgcga cgtcacgttc ccacgctacg
tcacacccct ctccaccaat caccgcccgc 34020cgcccccaac cctctccgcc
aatcaccacg ccacaaaagg ggcaataaaa gtgtgcggta 34080tattattgat gatg
34094244DNAPorcine Adenovirus Type 3 2gcggatcctt aattaacatc
atcaataata taccgcacac tttt 44332DNAPorcine Adenovirus Type 3
3cacctgcaga tacacccaca cacgtcatct cg 32432DNAPorcine Adenovirus
Type 3 4cacctgcagc ctcctgagtg tgaagagtgt cc 32520DNAPorcine
Adenovirus Type 3 5gactgacgcc ggcatgcaat 20627DNAPorcine Adenovirus
Type 3 6cggatcctga cgctacgagc ggttgta 27727DNAPorcine Adenovirus
Type 3 7cggatccata cgtacagatg aagtagc 27820DNAPorcine Adenovirus
Type 3 8tctgactgaa gccgacctgc 20918DNAPorcine Adenovirus Type 3
9ataggcgtat cacgaggc 181030DNAPorcine Adenovirus Type 3
10ctggactagt ctgttccgct gagagaaaac 301128DNAPorcine Adenovirus Type
3 11gtggactagt ctcatgcagc gaacaacc 281220DNAPorcine Adenovirus Type
3 12gtactatcac cttcctaagg 201320DNAPorcine Adenovirus Type 3
13acagtaatga ggaggatatc 201429DNAPorcine Adenovirus Type 3
14taggactagt cccacagaaa aagaaaagg 291528DNAPorcine Adenovirus Type
3 15atggactagt cttctggtgc cgccacta 281619DNAPorcine Adenovirus Type
3 16cctaatctgc tcaaagctg 191724DNAPorcine Adenovirus Type 3
17cgggatccgg ccgctgctgc agct 241825DNAPorcine Adenovirus Type 3
18gcgtcgactc aaaacaggct ctcat 25
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