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Here is a US patent for coronavirus. In my mind it means nothing without supplementary evidence
A
patent for corononavirus
Coronavirus
The present invention
provides a live, attenuated coronavirus comprising a variant
replicase gene encoding polyproteins comprising a mutation in one or
more of non-structural protein(s) (nsp)-10, nsp-14, nsp-15 or
nsp-16. The coronavirus may be used as a vaccine for treating and/or
preventing a disease, such as infectious bronchitis, in a subject.
Latest THE PIRBRIGHT INSTITUTE Patents:
Description
FIELD
OF THE INVENTION
The
present invention relates to an attenuated coronavirus comprising a
variant replicase gene, which causes the virus to have reduced
pathogenicity. The present invention also relates to the use of such
a coronavirus in a vaccine to prevent and/or treat a disease.
BACKGROUND
TO THE INVENTION
Avian
infectious bronchitis virus (IBV), the aetiological agent of
infectious bronchitis (IB), is a highly infectious and contagious
pathogen of domestic fowl that replicates primarily in the
respiratory tract but also in epithelial cells of the gut, kidney
and oviduct. IBV is a member of the Order Nidovirales, Family
Coronaviridae, Subfamily Corona virinae and Genus Gammacoronavirus;
genetically very similar coronaviruses cause disease in turkeys,
guinea fowl and pheasants.
Clinical
signs of IB include sneezing, tracheal rales, nasal discharge and
wheezing. Meat-type birds have reduced weight gain, whilst
egg-laying birds lay fewer eggs and produce poor quality eggs. The
respiratory infection predisposes chickens to secondary bacterial
infections which can be fatal in chicks. The virus can also cause
permanent damage to the oviduct, especially in chicks, leading to
reduced egg production and quality; and kidney, sometimes leading to
kidney disease which can be fatal.
IBV
has been reported to be responsible for more economic loss to the
poultry industry than any other infectious disease. Although live
attenuated vaccines and inactivated vaccines are universally used in
the control of IBV, the protection gained by use of vaccination can
be lost either due to vaccine breakdown or the introduction of a new
IBV serotype that is not related to the vaccine used, posing a risk
to the poultry industry.
Further,
there is a need in the industry to develop vaccines which are
suitable for use in ovo, in order to improve the efficiency and
cost-effectiveness of vaccination programmes. A major challenge
associated with in ovo vaccination is that the virus must be capable
of replicating in the presence of maternally-derived antibodies
against the virus, without being pathogenic to the embryo. Current
IBV vaccines are derived following multiple passage in embryonated
eggs, this results in viruses with reduced pathogenicity for
chickens, so that they can be used as live attenuated vaccines.
However such viruses almost always show an increased virulence to
embryos and therefore cannot be used for in ova vaccination as they
cause reduced hatchability. A 70% reduction in hatchability is seen
in some cases.
Attenuation
following multiple passage in embryonated eggs also suffers from
other disadvantages. It is an empirical method, as attenuation of
the viruses is random and will differ every time the virus is
passaged, so passage of the same virus through a different series of
eggs for attenuation purposes will lead to a different set of
mutations leading to attenuation. There are also efficacy problems
associated with the process: some mutations will affect the
replication of the virus and some of the mutations may make the
virus too attenuated. Mutations can also occur in the S gene which
may also affect immunogenicity so that the desired immune response
is affected and the potential vaccine may not protect against the
required serotype. In addition there are problems associated with
reversion to virulence and stability of vaccines.
It
is important that new and safer vaccines are developed for the
control of IBV. Thus there is a need for IBV vaccines which are not
associated with these issues, in particular vaccines which may be
used for in ovo vaccination.
SUMMARY
OF ASPECTS OF THE INVENTION
The
present inventors have used a reverse genetics approach in order to
rationally attenuate IBV. This approach is much more controllable
than random attenuation following multiple passages in embryonated
eggs because the position of each mutation is known and its effect
on the virus, i.e. the reason for attenuation, can be derived.
Using
their reverse genetics approach, the present inventors have
identified various mutations which cause the virus to have reduced
levels of pathogenicity. The levels of pathogenicity may be reduced
such that when the virus is administered to an embryonated egg, it
is capable of replicating without being pathogenic to the embryo.
Such viruses may be suitable for in ovo vaccination, which is a
significant advantage and has improvement over attenuated IBV
vaccines produced following multiple passage in embryonated eggs.
Thus
in a first aspect, the present invention provides a live, attenuated
coronavirus comprising a variant replicase gene encoding
polyproteins comprising a mutation in one or more of non-structural
protein(s) (nsp)-10, nsp-14, nsp-15 or nsp-16.
The
variant replicase gene may encode a protein comprising one or more
amino acid mutations selected from the list of:
The
replicase gene may encode a protein comprising the amino acid
mutation Pro to Leu at position 85 of SEQ ID NO: 6.
The
replicase gene may encode a protein comprising the amino acid
mutations Val to Leu at position 393 of SEQ ID NO: 7; Leu to Ile at
position 183 of SEQ ID NO: 8; and Val to Ile at position 209 of SEQ
ID NO: 9.
The
replicase gene may encodes a protein comprising the amino acid
mutations Pro to Leu at position 85 of SEQ ID NO: 6; Val to Leu at
position 393 of SEQ ID NO:7; Leu to Ile at position 183 of SEQ ID
NO:8; and Val to Ile at position 209 of SEQ ID NO: 9.
The
coronavirus may comprise an S protein at least part of which is from
an IBV serotype other than M41.
The
coronavirus according to the first aspect of the invention has
reduced pathogenicity compared to a coronavirus expressing a
corresponding wild-type replicase, such that when the virus is
administered to an embryonated egg, it is capable of replicating
without being pathogenic to the embryo.
In
a second aspect, the present invention provides a variant replicase
gene as defined in connection with the first aspect of the
invention.
In
a third aspect, the present invention provides a protein encoded by
a variant coronavirus replicase gene according to the second aspect
of the invention.
In
a fourth aspect, the present invention provides a plasmid comprising
a replicase gene according to the second aspect of the invention.
In
a fifth aspect, the present invention provides a method for making
the coronavirus according to the first aspect of the invention which
comprises the following steps:
In
a sixth aspect, the present invention provides a cell capable of
producing a coronavirus according to the first aspect of the
invention.
In
a seventh aspect, the present invention provides a vaccine
comprising a coronavirus according to the first aspect of the
invention and a pharmaceutically acceptable carrier.
In
an eighth aspect, the present invention provides a method for
treating and/or preventing a disease in a subject which comprises
the step of administering a vaccine according to the seventh aspect
of the invention to the subject.
The
method of administration of the vaccine may be selected from the
group consisting of; eye drop administration, intranasal
administration, drinking water administration, post-hatch injection
and in ovo injection.
The
present invention also provides a method for producing a vaccine
according to the seventh aspect of the invention, which comprises
the step of infecting a cell according to the sixth aspect of the
invention with a coronavirus according to the first aspect of the
invention.
class="heading-4"
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1.375rem;"DESCRIPTION
OF THE FIGURES
FIG.
2—Clinical signs, snicking and wheezing, associated with M41-R-6
and M41-R-12 compared to M41-CK (M41 EP4) and Beau-R (Bars show
mock, Beau-R, M41-R 6, M41-R 12, M41-CK EP4 from left to right of
each timepoint).
FIG.
3—Ciliary activity of the viruses in tracheal rings isolated from
tracheas taken from infected chicks. 100% ciliary activity indicates
no effect by the virus; apathogenic, 0% activity indicates complete
loss of ciliary activity, complete ciliostasis, indicating the virus
is pathogenic (Bars show mock, Beau-R, M41-R 6, M41-R 12, M41-CK EP4
from left to right of each timepoint).
FIG.
4—Clinical signs, snicking, associated with M41R-nsp10rep and
M41R-nsp14,15,16rep compared to M41-R-12 and M41-CK (M41 EP5) (Bars
show mock, M41-R12; M41R-nsp10rep; M41R-nsp14,15,16rep and M41-CK
EP5 from left to right of each timepoint).
FIG.
5—Ciliary activity of M41R-nsp10rep and M41R-nsp14,15,16rep
compared to M41-R-12 and M41-CK in tracheal rings isolated from
tracheas taken from infected chicks (Bars show mock; M41-R12;
M41R-nsp10rep; M41R-nsp14,15,16rep and M41-CK EP5 from left to right
of each timepoint).
FIG.
6—Clinical signs, snicking, associated with M41R-nsp10, 15rep,
M41R-nsp10, 14, 15rep, M41R-nsp10, 14, 16rep, M41R-nsp10, 15, 16rep
and M41-K compared to M41-CK (Bars show mock, M41R-nsp10,15rep1;
M41R-nsp10,14,16rep4; M41R-nsp10,15,16rep8; M41R-nsp10,14,15rep10;
M41-K6 and M41-CK EP4 from left to right of each timepoint).
FIG.
7—Clinical signs, wheezing, associated with M41R-nsp10, 15rep,
M41R-nsp10, 14, 15rep, M41R-nsp10, 14, 16rep, M41R-nsp10, 15, 16rep
and M41-K compared to M41-CK (Bars show mock, M41R-nsp10,15rep1;
M14R-nsp10,14,16rep4; M41R-nsp10,15,16rep8; M41R-nsp10,14,15rep10;
M41-K6 and M41-CK EP4 from left to right of each timepoint).
FIG.
8—Ciliary activity of M41R-nsp10, 15rep, M41R-nsp10, 14, 15rep,
M41R-nsp10, 14, 16rep, M41R-nsp10, 15, 16rep and M41-K compared to
M41-CK in tracheal rings isolated from tracheas taken from infected
chicks (Bars show mock, M41R-nsp10,15rep1; M41R-nsp10,14,16rep4;
M41R-nsp10,15,16rep8; M41R-nsp10,14,15rep10; M41-K6 and M41-CK EP4
from left to right of each timepoint).
FIG.
9—Growth kinetics of rIBVs compared to M41-CK on CK cells. FIG.
9A shows the results for M41-R and M41-K. FIG. 9B shows
the results for M41-nsp10 rep; M41R-nsp14, 15, 16 rep; M41R-nsp10,
15 rep; M41R-nsp10, 15, 16 rep; M41R-nsp10, 14, 15 rep; and
M41R-nsp10, 14, 16.
FIG.
11—A) Snicking; B) Respiratory symptoms (wheezing and rales
combined) and C) Ciliary activity of rIBV M41R-nsp 10,14 rep and
rIBV M41R-nsp 10,16 rep compared to M41-CK (Bars show mock,
M41R-nsp10,14rep; M41R-nsp10,16rep and M41-K from left to right of
each timepoint).
DETAILED
DESCRIPTION
The
present invention provides a coronavirus comprising a variant
replicase gene which, when expressed in the coronavirus, causes the
virus to have reduced pathogenicity compared to a corresponding
coronavirus which comprises the wild-type replicase gene.
Gammacoronavirus is
a genus of animal virus belonging to the family Coronaviridae.
Coronaviruses are enveloped viruses with a positive-sense
single-stranded RNA genome and a helical symmetry.
The
genomic size of coronaviruses ranges from approximately 27 to 32
kilobases, which is the longest size for any known RNA virus.
Coronaviruses
primarily infect the upper respiratory or gastrointestinal tract of
mammals and birds. Five to six different currently known strains of
coronaviruses infect humans. The most publicized human coronavirus,
SARS-CoV which causes severe acute respiratory syndrome (SARS), has
a unique pathogenesis because it causes both upper and lower
respiratory tract infections and can also cause gastroenteritis.
Middle East respiratory syndrome coronavirus (MERS-CoV) also causes
a lower respiratory tract infection in humans. Coronaviruses are
believed to cause a significant percentage of all common colds in
human adults.
Coronaviruses
also cause a range of diseases in livestock animals and domesticated
pets, some of which can be serious and are a threat to the farming
industry. Economically significant coronaviruses of livestock
animals include infectious bronchitis virus (IBV) which mainly
causes respiratory disease in chickens and seriously affects the
poultry industry worldwide; porcine coronavirus (transmissible
gastroenteritis, TGE) and bovine coronavirus, which both result in
diarrhoea in young animals. Feline coronavirus has two forms, feline
enteric coronavirus is a pathogen of minor clinical significance,
but spontaneous mutation of this virus can result in feline
infectious peritonitis (FIP), a disease associated with high
mortality.
There
are also two types of canine coronavirus (CCoV), one that causes
mild gastrointestinal disease and one that has been found to cause
respiratory disease. Mouse hepatitis virus (MHV) is a coronavirus
that causes an epidemic murine illness with high mortality,
especially among colonies of laboratory mice.
The
variant replicase gene of the coronavirus of the present invention
may be derived from an alphacoronavirus such as TGEV; a
betacoronavirus such as MHV; or a gammacoronavirus such
as IBV.
As
used herein the term “derived from” means that the replicase
gene comprises substantially the same nucleotide sequence as the
wild-type replicase gene of the relevant coronavirus. For example,
the variant replicase gene of the present invention may have up to
80%, 85%, 90%, 95%, 98% or 99% identity with the wild type replicase
sequence. The variant coronavirus replicase gene encodes a protein
comprising a mutation in one or more of non-structural protein
(nsp)-10, nsp-14, nsp-15 or nsp-16 when compared to the wild-type
sequence of the non-structural protein.
Avian
infectious bronchitis (IB) is an acute and highly contagious
respiratory disease of chickens which causes significant economic
losses. The disease is characterized by respiratory signs including
gasping, coughing, sneezing, tracheal rales, and nasal discharge. In
young chickens, severe respiratory distress may occur. In layers,
respiratory distress, nephritis, decrease in egg production, and
loss of internal egg quality and egg shell quality are common.
In
broilers, coughing and rattling are common clinical signs, rapidly
spreading in all the birds of the premises. Morbidity is 100% in
non-vaccinated flocks. Mortality varies depending on age, virus
strain, and secondary infections but may be up to 60% in
non-vaccinated flocks.
The
first IBV serotype to be identified was Massachusetts, but in the
United States several serotypes, including Arkansas and Delaware,
are currently circulating, in addition to the originally identified
Massachusetts type.
The
IBV strain Beaudette was derived following at least 150 passages in
chick embryos. IBV Beaudette is no longer pathogenic for hatched
chickens but rapidly kills embryos.
H120
is a commercial live attenuated IBV Massachusetts serotype vaccine
strain, attenuated by approximately 120 passages in embryonated
chicken eggs. H52 is another Massachusetts vaccine, and represents
an earlier and slightly more pathogenic passage virus (passage 52)
during the development of H120. Vaccines based on H120 are commonly
used.
IB
QX is a virulent field isolate of IBV. It is sometimes known as
“Chinese QX” as it was originally isolated following outbreaks
of disease in the Qingdao region in China in the mid 1990s. Since
that time the virus has crept towards Europe. From 2004, severe egg
production issues have been identified with a very similar virus in
parts of Western Europe, predominantly in the Netherlands, but also
reported from Germany, France, Belgium, Denmark and in the UK.
The
virus isolated from the Dutch cases was identified by the Dutch
Research Institute at Deventer as a new strain that they called
D388. The Chinese connection came from further tests which showed
that the virus was 99% similar to the Chinese QX viruses. A live
attenuated QX-like IBV vaccine strain has now been developed.
IBV
is an enveloped virus that replicates in the cell cytoplasm and
contains an non-segmented, single-stranded, positive sense RNA
genome. IBV has a 27.6 kb RNA genome and like all coronaviruses
contains the four structural proteins; spike glycoprotein (S), small
membrane protein (E), integral membrane protein (M) and nucleocapsid
protein (N) which interacts with the genomic RNA.
The
genome is organised in the following manner: 5′UTR—polymerase
(replicase) gene—structural protein genes (S-E-M-N)—UTR 3′;
where the UTR are untranslated regions (each ˜500 nucleotides in
IBV).
The
lipid envelope contains three membrane proteins: S, M and E. The IBV
S protein is a type I glycoprotein which oligomerizes in the
endoplasmic reticulum and is assembled into homotrimer inserted in
the virion membrane via the transmembrane domain and is associated
through non-covalent interactions with the M protein. Following
incorporation into coronavirus particles, the S protein is
responsible for binding to the target cell receptor and fusion of
the viral and cellular membranes. The S glycoprotein consists of
four domains: a signal sequence that is cleaved during synthesis;
the ectodomain, which is present on the outside of the virion
particle; the transmembrane region responsible for anchoring the S
protein into the lipid bilayer of the virion particle; and the
cytoplasmic tail.
All
coronaviruses also encode a set of accessory protein genes of
unknown function that are not required for replication in vitro, but
may play a role in pathogenesis. IBV encodes two accessory genes,
genes 3 and 5, which both express two accessory proteins 3a, 3b and
5a, 5b, respectively.
The
variant replicase gene of the coronavirus of the present invention
may be derived from an IBV. For example the IBV may be IBV
Beaudette, H120, H52, IB QX, D388 or M41.
The
IBV may be IBV M41. M41 is a prototypic Massachusetts serotype that
was isolated in the USA in 1941. It is an isolate used in many labs
throughout the world as a pathogenic lab stain and can be obtained
from ATCC (VR-21™). Attenuated variants are also used by several
vaccine producers as IBV vaccines against Massachusetts serotypes
causing problems in the field. The present inventors chose to use
this strain as they had worked for many years on this virus, and
because the sequence of the complete virus genome is available. The
M41 isolate, M41-CK, used by the present inventors was adapted to
grow in primary chick kidney (CK) cells and was therefore deemed
amenable for recovery as an infectious virus from a cDNA of the
complete genome. It is representative of a pathogenic IBV and
therefore can be analysed for mutations that cause either loss or
reduction in pathogenicity.
colname="1"
colwidth="259pt" align="left" style="box-sizing:
border-box;"colname="2" colwidth="182pt"
align="left" style="box-sizing:
border-box;"IBV M41-CK Sequence SEQ ID NO: 1 ACTTAAGATAGATATTAATATATATCTATCACACTAGCCTTGCGCTAGATTTCCAACTTA ACAAAACGGACTTAAATACCTACAGCTGGTCCTCATAGGTGTTCCATTGCAGTGCACTTT AGTGCCCTGGATGGCACCTGGCCACCTGTCAGGTTTTTGTTATTAAAATCTTATTGTTGC TGGTATCACTGCTTGTTTTGCCGTGTCTCACTTTATACATCCGTTGCTTGGGCTACCTAG TATCCAGCGTCCTACGGGCGCCGTGGCTGGTTCGAGTGCGAAGAACCTCTGGTTCATCTA GCGGTAGGCGGGTGTGTGGAAGTAGCACTTCAGACGTACCGGTTCTGTTGTGTGAAATAC GGGGTCACCTCCCCCCACATACCTCTAAGGGCTTTTGAGCCTAGCGTTGGGCTACGTTCT CGCATAAGGTCGGCTATACGACGTTTGTAGGGGGTAGTGCCAAACAACCCCTGAGGTGAC AGGTTCTGGTGGTGTTTAGTGAGCAGACATACAATAGACAGTGACAACATGGCTTCAAGC CTAAAACAGGGAGTATCTGCGAAACTAAGGGATGTCATTGTTGTATCCAAAGAGATTGCT GAACAACTTTGTGACGCTTTGTTTTTCTATACGTCACACAACCCTAAGGATTACGCTGAT GCTTTTGCAGTTAGGCAGAAGTTTGATCGTAATCTGCAGACTGGGAAACAGTTCAAATTT GAAACTGTGTGTGGTCTCTTCCTCTTGAAGGGAGTTGACAAAATAACACCTGGCGTCCCA GCAAAAGTCTTAAAAGCCACTTCTAAGTTGGCAGATTTAGAAGACATCTTTGGTGTCTCT CCCTTTGCAAGAAAATATCGTGAACTTTTGAAGACAGCATGCCAGTGGTCTCTTACTGTA GAAACACTGGATGCTCGTGCACAAACTCTTGATGAAATTTTTGACCCTACTGAAATACTT TGGCTTCAGGTGGCAGCAAAAATCCAAGTTTCGGCTATGGCGATGCGCAGGCTTGTTGGA GAAGTAACTGCAAAAGTCATGGATGCTTTGGGCTCAAATATGAGTGCTCTTTTCCAGATT TTTAAACAACAAATAGTCAGAATTTTTCAAAAAGCGCTGGCTATTTTTGAGAATGTGAGT GAATTACCACAGCGTATTGCAGCACTTAAGATGGCTTTTGCTAAGTGTGCCAAGTCCATT ACTGTTGTGGTTATGGAGAGGACTCTAGTTGTTAGAGAGTTCGCAGGAACTTGTCTTGCA AGCATTAATGGTGCTGTTGCAAAATTCTTTGAAGAACTCCCAAATGGTTTCATGGGTGCT AAAATTTTCACTACACTTGCCTTCTTTAGGGAGGCTGCAGTGAAAATTGTGGATAACATA CCAAATGCACCGAGAGGCACTAAAGGGTTTGAAGTCGTTGGTAATGCCAAAGGTACACAA GTTGTTGTGCGTGGCATGGGAAATGACTTAACACTGGTTGAGCAAAAAGCTGAAATTGCT GTGGAGTCAGAAGGTTGGTCTGCAATTTTGGGTGGACATCTTTGCTATGTCTTTAAGAGT GGTGATCGCTTTTACGCGGCACCTCTTTCAGGAAATTTTGCATTGCATGATGTGCATTGT TGTGAGCGTGTTGTCTGTCTTTCTGATGGTGTAACACCGGAGATAAATGATGGACTTATT CTTGCAGCAATCTACTCTTCTTTTAGTGTCGCAGAACTTGTGGCAGCCATTAAAAGGGGT GAACCATTTAAGTTTCTGGGTCATAAATTTGTGTATGCAAAGGATGCAGCAGTTTCTTTT ACATTAGCGAAGGCTGCTACTATTGCAGATGTTTTGAAGCTGTTTCAATCAGCGCGTGTG AAAGTAGAAGATGTTTGGTCTTCACTTACTGAAAAGTCTTTTGAATTCTGGAGGCTTGCA TATGGAAAAGTGCGTAATCTCGAAGAATTTGTTAAGACTTGTTTTTGTAAGGCTCAAATG GCGATTGTGATTTTAGCGACAGTGCTTGGAGAGGGCATTTGGCATCTTGTTTCGCAAGTC ATCTATAAAGTAGGTGGTCTTTTTACTAAAGTTGTTGACTTTTGTGAAAAATATTGGAAA GGTTTTTGTGCACAGTTGAAAAGAGCTAAGCTCATTGTCACTGAAACCCTCTGTGTTTTG AAAGGAGTTGCACAGCATTGTTTTCAACTATTGCTGGATGCAATACAGTTTATGTATAAA AGTTTTAAGAAGTGTGCACTTGGTAGAATCCATGGAGACTTGCTCTTCTGGAAAGGAGGT GTGCACAAAATTATTCAAGAGGGCGATGAAATTTGGTTTGAGGGCATTGATAGTATTGAT GTTGAAGATCTGGGTGTTGTTCAAGAAAAATTGATTGATTTTGATGTTTGTGATAATGTG ACACTTCCAGAGAACCAACCCGGTCATATGGTTCAAATCGAGGATGACGGAAAGAACTAC ATGTTCTTCCGCTTCAAAAAGGATGAGAACATTTATTATACACCAATGTCACAGCTTGGT GCTATTAATGTGGTTTGCAAAGCAGGCGGTAAAACTGTCACCTTTGGAGAAACTACTGTG CAAGAAATACCACCACCTGATGTTGTGTTTATTAAGGTTAGCATTGAGTGTTGTGGTGAA CCATGGAATACAATCTTCAAAAAGGCTTATAAGGAGCCCATTGAAGTAGAGACAGACCTC ACAGTTGAACAATTGCTCTCTGTGGTCTATGAGAAAATGTGTGATGATCTCAAGCTGTTT CCGGAGGCTCCAGAACCACCACCATTTGAGAATGTCACACTTGTTGATAAGAATGGTAAA GATTTGGATTGCATAAAATCATGCCATCTGATCTATCGTGATTATGAGAGCGATGATGAC ATCGAGGAAGAAGATGCAGAAGAATGTGACACGGATTCAGGTGATGCTGAGGAGTGTGAC ACTAATTCAGAATGTGAAGAAGAAGATGAGGATACTAAAGTGTTGGCTCTTATACAAGAC CCGGCAAGTAACAAATATCCTCTGCCTCTTGATGATGATTATAGCGTCTACAATGGATGT ATTGTTCATAAGGACGCTCTCGATGTTGTGAATTTACCATCTGGTGAAGAAACCTTTGTT GTCAATAACTGCTTTGAAGGGGCTGTTAAAGCTCTTCCGCAGAAAGTTATTGATGTTCTA GGTGACTGGGGTGAGGCTGTTGATGCGCAAGAACAATTGTGTCAACAAGAATCAACTCGG GTCATATCTGAGAAATCAGTTGAGGGTTTTACTGGTAGTTGTGATGCAATGGCTGAACAA GCTATTGTTGAAGAGCAGGAAATAGTACCTGTTGTTGAACAAAGTCAGGATGTAGTTGTT TTTACACCTGCAGACCTAGAAGTTGTTAAAGAAACAGCAGAAGAGGTTGATGAGTTTATT CTCATTTCTGCTGTCCCTAAAGAAGAAGTTGTGTCTCAGGAGAAAGAGGAGCCACAGGTT GAGCAAGAGCCTACCCTAGTTGTTAAAGCACAACGTGAGAAGAAGGCTAAAAAGTTCAAA GTTAAACCAGCTACATGTGAAAAACCCAAATTTTTGGAGTACAAAACATGTGTGGGTGAT TTGGCTGTTGTAATTGCCAAAGCATTGGATGAGTTTAAAGAGTTCTGCATTGTAAACGCT GCAAATGAGCACATGTCGCATGGTGGTGGCGTTGCAAAGGCAATTGCAGACTTTTGTGGA CCGGACTTTGTTGAATATTGCGCGGACTATGTTAAGAAACATGGTCCACAGCAAAAACTT GTCACACCTTCATTTGTTAAAGGCATTCAATGTGTGAATAATGTTGTAGGACCTCGCCAT GGAGACAGCAACTTGCGTGAGAAGCTTGTTGCTGCTTACAAGAGTGTTCTTGTAGGTGGA GTGGTTAACTATGTTGTGCCAGTTCTCTCATCAGGGATTTTTGGTGTAGATTTTAAAATA TCAATAGATGCTATGCGCGAAGCTTTTAAAGGTTGTGCCATACGCGTTCTTTTATTTTCT CTGAGTCAAGAACACATCGATTATTTCGATGCAACTTGTAAGCAGAAGACAATTTATCTT ACGGAGGATGGTGTTAAATACCGCTCTGTTGTTTTAAAACCTGGTGATTCTTTGGGTCAA TTTGGACAGGTTTTTGCAAGAAATAAGGTAGTCTTTTCGGCTGATGATGTTGAGGATAAA GAAATCCTCTTTATACCCACAACTGACAAGACTATTCTTGAATATTATGGTTTAGATGCG CAAAAGTATGTAACATATTTGCAAACGCTTGCGCAGARATGGGATGTTCAATATAGAGAC AATTTTGTTATATTAGAGTGGCGTGACGGAAATTGCTGGATTAGTTCAGCAATAGTTCTC CTTCAAGCTGCTAAAATTAGATTTAAAGGTTTTCTTGCAGAAGCATGGGCTAAACTGTTG GGTGGAGATCCTACAGACTTTGTTGCCTGGTGTTATGCAAGTTGCAATGCTAAAGTAGGT GATTTTTCAGATGCTAATTGGCTTTTGGCCAATTTAGCAGAACATTTTGACGCAGATTAC ACAAATGCACTTCTTAAGAAGTGTGTGTCGTGCAATTGTGGTGTTAAGAGTTATGAACTT AGGGGTCTTGAAGCCTGTATTCAGCCAGTTCGAGCACCTAATCTTCTACATTTTAAAACG CAATATTCAAATTGCCCAACCTGTGGTGCAAGTAGTACGGATGAAGTAATAGAAGCTTCA TTACCGTACTTATTGCTTTTTGCTACTGATGGTCCTGCTACAGTTGATTGTGATGAAAAT GCTGTAGGGACTGTTGTTTTCATTGGCTCTACTAATAGTGGCCATTGTTATACACAAGCC GATGGTAAGGCTTTTGACAATCTTGCTAAGGATAGAAAATTTGGAAGGAAGTCGCCTTAC ATTACAGCAATGTATACACGTTTTTCTCTTAGGAGTGAAAATCCCCTACTTGTTGTTGAA CATAGTAAGGGTAAAGCTAAAGTAGTAAAAGAAGATGTTTCTAACCTTGCTACTAGTTCT AAAGCCAGTTTTGACGATCTTACTGACTTTGAACACTGGTATGATAGCAACATCTATGAG AGTCTTAAAGTGCAGGAGACACCTGATAATCTTGATGAATATGTGTCATTTACGACAAAG GAAGATTCTAAGTTGCCACTGACACTTAAAGTTAGAGGTATCAAATCAGTTGTTGACTTT AGGTCTAAGGATGGTTTTACTTATAAGTTAACACCTGATACTGATGAAAATTCAAAAACA CCAGTCTACTACCCAGTCTTGGATTCTATTAGTCTTAGGGCAATATGGGTTGAAGGCAGT GCTAATTTTGTTGTTGGGCATCCAAATTATTATAGTAAGTCTCTCCGAATTCCCACGTTT TGGGAAAATGCCGAGAGCTTTGTTAAAATGGGTTATAAAATTGATGGTGTAACTATGGGC CTTTGGCGTGCAGAACACCTTAATAAACCTAATTTGGAGAGAATTTTTAACATTGCTAAG AAAGCTATTGTTGGATCTAGTGTTGTTACTACGCAGTGTGGTAAAATACTAGTTAAAGCA GCTACATACGTTGCCGATAAAGTAGGTGATGGTGTAGTTCGCAATATTACAGATAGAATT AAGGGTCTTTGTGGATTCACACGTGGCCATTTTGAAAAGAAAATGTCCCTACAATTTCTA AAGACACTTGTGTTCTTTTTCTTTTATTTCTTAAAGGCTAGTGCTAAGAGTTTAGTTTCT AGCTATAAGATTGTGTTATGTAAGGTGGTGTTTGCTACCTTACTTATAGTGTGGTTTATA TACACAAGTAATCCAGTAGTGTTTACTGGAATACGTGTGCTAGACTTCCTATTTGAAGGT TCTTTATGTGGTCCTTATAATGACTACGGTAAAGATTCTTTTGATGTGTTACGGTATTGT GCAGGTGATTTTACTTGTCGTGTGTGTTTACATGATAGAGATTCACTTCATCTGTACAAA CATGCTTATAGCGTAGAACAAATTTATAAGGATGCAGCTTCTGGCATTAACTTTAATTGG AATTGGCTTTATTTGGTCTTTCTAATATTATTTGTTAAGCCAGTGGCAGGTTTTGTTATT ATTTGTTATTGTGTTAAGTATTTGGTATTGAGTTCAACTGTGTTGCAAACTGGTGTAGGT TTTCTAGATTGGTTTGTAAAAACAGTTTTTACCCATTTTAATTTTATGGGAGCGGGATTT TATTTCTGGCTCTTTTACAAGATATACGTACAAGTGCATCATATATTGTACTGTAAGGAT GTAACATGTGAAGTGTGCAAGAGAGTTGCACGCAGCAACAGGCAAGAGGTTAGCGTTGTA GTTGGTGGACGCAAGCAAATAGTGCATGTTTACACTAATTCTGGCTATAACTTTTGTAAG AGACATAATTGGTATTGTAGAAATTGTGATGATTATGGTCACCAAAATACATTTATGTCC CCTGAAGTTGCTGGCGAGCTTTCTGAAAAGCTTAAGCGCCATGTTAAACCTACAGCATAT GCTTACCACGTTGTGTATGAGGCATGCGTGGTTGATGATTTTGTTAATTTAAAATATAAG GCTGCAATTGCTGGTAAGGATAATGCATCTTCTGCTGTTAAGTGTTTCAGTGTTACAGAT TTTTTAAAGAAAGCTGTTTTTCTTAAGGAGGCATTGAAATGTGAACAAATATCTAATGAT GGTTTTATAGTGTGTAATACACAGAGTGCGCATGCACTAGAGGAAGCAAAGAATGCAGCC GTCTATTATGCGCAATATCTGTGTAAGCCAATACTTATACTTGACCAGGCACTTTATGAG CAATTAATAGTAGAGCCTGTGTCTAAGAGTGTTATAGATAAAGTGTGTAGCATTTTGTCT AATATAATATCTGTAGATACTGCAGCTTTAAATTATAAGGCAGGCACACTTCGTGATGCT CTGCTTTCTATTACTAAAGACGAAGAAGCCGTAGATATGGCTATCTTCTGCCACAATCAT GAAGTGGAATACACTGGTGACGGTTTTACTAATGTGATACCGTCATATGGTATGGACACT GATAAGTTGACACCTCGTGATAGAGGGTTTTTGATAAATGCAGATGCTTCTATTGCTAAT TTAAGAGTCAAAAATGCTCCTCCGGTAGTATGGAAGTTTTCTGATCTTATTAAATTGTCT GACAGTTGCCTTAAATATTTAATTTCAGCTACTGTCAAGTCAGGAGGTCGTTTCTTTATA ACAAAGTCTGGTGCTAAACAAGTTATTTCTTGTCATACCCAGAAACTGTTGGTAGAGAAA AAGGCAGGTGGTGTTATTAATAACACTTTTAAATGGTTTATGAGTTGTTTTAAATGGCTT TTTGTCTTTTATATACTTTTTACAGCATGTTGTTTGGGTTACTACTATATGGAGATGAAT AAAAGTTTTGTTCACCCCATGTATGATGTAAACTCCACACTGCATGTTGAAGGGTTCAAA GTTATAGACAAAGGTGTTATTAGAGAGATTGTGTCAGAAGATAATTGTTTCTCTAATAAG TTTGTTAATTTTGACGCCTTTTGGGGTAAATCATATGAAAATAATAAAAACTGTCCAATT GTTACAGTTGTTATAGATGGTGACGGGACAGTAGCTGTTGGTGTTCCTGGTTTTGTATCA TGGGTTATGGATGGTGTTATGTTTGTGCATATGACACAGACTGATCGTAGACCTTGGTAC ATTCCTACCTGGTTTAATAGAGAAATTGTTGGTTACACTCAGGATTCAATTATCACTGAG GGTAGTTTTTATACATCTATAGCATTATTTTCTGCTAGATGTTTATATTTAACAGCCAGC AATACACCTCAATTGTATTGTTTTAATGGCGACAATGATGCACCTGGAGCCTTACCATTT GGTAGTATTATTCCTCATAGAGTATACTTCCAACCTAATGGTGTTAGGCTTATAGTTCCA CAACAAATACTGCATACACCCTACATAGTGAAGTTTGTTTCAGACAGCTATTGTAGAGGT AGTGTATGTGAGTATACTAAACCAGGTTACTGTGTGTCACTAGACTCCCAATGGGTTTTG TTTAATGATGAATACATTAGTAAACCTGGCGTTTTCTGTGGTTCTACTGTTAGAGAACTT ATGTTTAATATGGTTAGTACATTCTTTACTGGTGTCAACCCTAATATTTATATTCAGCTA GCAACTATGTTTTTAATACTAGTTGTTATTGTGTTAATTTTTGCAATGGTTATAAAGTTT CAAGGTGTTTTTAAAGCTTATGCGACCATTGTGTTTACAATAATGTTAGTTTGGGTTATT AATGCATTTGTTTTGTGTGTACATAGTTATAATAGTGTTTTAGCTGTTATATTATTAGTA CTCTATTGCTATGCATCATTGGTTACAAGTCGCAATACTGCTATAATAATGCATTGTTGG CTTGTTTTTACCTTTGGTTTAATAGTACCCACATGGTTGGCTTGTTGCTATCTGGGATTT ATTCTTTATATGTACACACCGTTGGTTTTCTGGTGTTACGGTACTACTAAAAATACTCGT AAGTTGTATGATGGCAACGAGTTTGTTGGTAATTATGACCTTGCTGCGAAGAGCACTTTT GTTATTCGTGGTACTGAATTTGTTAAGCTTACGAATGAGATAGGTGATAAATTTGAAGCC TATCTTTCTGCGTATGCTAGACTTAAATACTATTCAGGCACTGGTAGTGAGCAAGATTAC TTGCAAGCTTGTCGTGCATGGTTAGCTTATGCTTTGGACCAATATAGAAATAGTGGTGTT GAGGTTGTTTATACCCCACCGCGTTACTCTATTGGTGTTAGTAGACTACACGCTGGTTTT AAAAAACTAGTTTCTCCTAGTAGTGCTGTTGAGAAGTGCATTGTTAGTGTCTCTTATAGA GGCAATAATCTTAATGGACTGTGGCTGGGTGATTCTATTTACTGCCCACGCCATGTGTTA GGTAAGTTTAGTGGTGACCAGTGGGGTGACGTACTAAACCTTGCTAATAATCATGAGTTT GAAGTTGTAACTCAAAATGGTGTTACTTTGAATGTTGTCAGCAGGCGGCTTAAAGGAGCA GTTTTAATTTTACAAACTGCAGTTGCCAATGCTGAAACTCCTAAGTATAAGTTTGTTAAA GCTAATTGTGGTGATAGTTTCACTATAGCTTGTTCTTATGGTGGTACAGTTATAGGACTT TACCCTGTCACTATGCGTTCTAATGGTACTATTAGAGCATCTTTCCTAGCAGGAGCCTGT GGCTCAGTTGGTTTTAATATAGAAAAGGGTGTAGTTAATTTCTTTTATATGCACCATCTT GAGTTACCTAATGCATTACACACTGGAACTGACCTAATGGGTGAGTTTTATGGTGGTTAT GTAGATGAAGAGGTTGCGCAAAGAGTGCCACCAGATAATCTAGTTACTAACAATATTGTA GCATGGCTCTATGGGGCAATTATTAGTGTTAAAGAAAGTAGTTTTTCACAACCTAAATGG TTGGAGAGTACTACTGTTTCTATTGAAGATTACAATAGGTGGGCTAGTGATAATGGTTTT ACTCCATTTTCCACTAGTACTGCTATTACTAAATTAAGTGCTATAACTGGGGTTGATGTT TGTAAACTCCTTCGCACTATTATGGTAAAAAGTGCTCAATGGGGTAGTGATCCCATTTTA GGACAATATAATTTTGAAGACGAATTGACACCAGAATCTGTATTTAATCAAGTTGGTGGT GTTAGGTTACAGTCTTCTTTTGTAAGAAAAGCTACATCTTGGTTTTGGAGTAGATGTGTA TTAGCTTGCTTCTTGTTTGTGTTGTGTGCTATTGTCTTATTTACGGCAGTGCCACTTAAG TTTTATGTACATGCAGCTGTTATTTTGTTGATGGCTGTGCTCTTTATTTCTTTTACTGTT AAACATGTTATGGCATACATGGACACTTTCCTATTGCCTACATTGATTACAGTTATTATT GGAGTTTGTGCTGAAGTCCCTTTCATATACAATACTCTAATTAGTCAAGTTGTTATTTTC TTAAGCCAATGGTATGATCCTGTAGTCTTTGATACTATGGTACCATGGATGTTATTGCCA TTAGTGTTGTACACTGCTTTTAAGTGTGTACAAGGCTGCTATATGAATTCTTTCAATACT TCTTTGTTAATGCTGTATCAGTTTATGAAGTTAGGTTTTGTTATTTACACCTCTTGAAAC ACTCTTACTGCATATACAGAAGGTAATTGGGAGTTATTCTTTGAGTTGGTTCACACTATT GTGTTGGCTAATGTTAGTAGTAATTCCTTAATTGGTTTAATTGTTTTTAAGTGTGCTAAG TGGATTTTATATTATTGCAATGCAACATACTTTAATAATTATGTGTTAATGGCAGTCATG GTTAATGGCATAGGCTGGCTTTGCACCTGTTACTTTGGATTGTATTGGTGGGTTAATAAA GTTTTTGGTTTAACCTTAGGTAAATACAATTTTAAAGTTTCAGTAGATCAATATAGGTAT ATGTGTTTGCATAAGGTAAATCCACCTAAAACTGTGTGGGAGGTCTTTACTACAAATATA CTTATACAAGGAATTGGAGGCGATCGTGTGTTGCCTATAGCTACAGTGCAATCTAAATTG AGTGATGTAAAGTGTACAACTGTTGTTTTAATGCAGCTTTTGACTAAGCTTAATGTTGAA GCAAATTCAAAAATGCATGCTTATCTTGTTGAGTTACACAATAAAATCCTCGCATCTGAT GATGTTGGAGAGTGCATGGATAATTTATTGGGTATGCTTATAACACTATTTTGTATAGAT TCTACTATTGATTTGGGTGAGTATTGTGATGATATACTTAAGAGGTCAACTGTATTACAA TCGGTTACTCAAGAGTTTTCGCACATACCCTCGTATGCTGAATATGAAAGAGCTAAGAGT ATTTATGAAAAGGTTTTAGCCGATTCTAAAAATGGTGGTGTAACACAGCAAGAGCTTGCT GCATATCGTAAAGCTGCCAATATTGCAAAGTCAGTTTTTGATAGAGACTTGGCTGTTCAA AAGAAGTTAGATAGCATGGCAGAACGTGCTATGACAACAATGTATAAAGAGGCGCGTGTA ACTGATAGAAGAGCAAAATTAGTTTCATCATTACATGCACTACTTTTTTCAATGCTTAAG AAAATAGATTCTGAGAAGCTTAATGTCTTATTTGACCAGGCGAATAGTGGTGTTGTACCC CTAGCAACTGTTCCAATTGTTTGTAGTAATAAGCTTACCCTTGTTATACCAGACCCAGAG ACGTGGGTCAAGTGTGTGGAGGGTGTGCATGTTACATATTCAACAGTTGTTTGGAATATA GACTGTGTTACTGATGCCGATGGCACAGAGTTACACCCCACTTCTACAGGTAGTGGATTG ACTTACTGTATAAGTGGTGATAATATAGCATGGCCTTTAAAGGTTAACTTGACTAGGAAT GGGCATAATAAGGTTGATGTTGCCTTGCAAAATAATGAGCTTATGCCTCACGGTGTAAAG ACAAAGGCTTGCGTAGCAGGTGTAGATCAAGCACATTGTAGCGTTGAGTCTAAATGTTAT TATACAAGTATTAGTGGCAGTTCAGTTGTAGCTGCTATTACCTCTTCAAATCCTAATCTG AAAGTAGCCTCTTTTTTGAATGAGGCAGGTAATCAGATTTATGTAGACTTAGACCGAGCA TGTAAATTTGGTATGAAAGTGGGTGATAAGGTTGAAGTTGTTTACCTGTATTTTATAAAA AATACGAGGTCTATTGTAAGAGGTATGGTACTTGGTGCTATATCTAATGTTGTTGTGTTA CAATCTAAAGGTCATGAGACAGAGGAAGTGGATGCTGTAGGCATTCTCTCACTTTGTTCT TTTGCAGTAGATCCTGCGGATACATATTGTAAATATGTGGCAGCAGGTAATCAACCTTTA GGTAACTGTGTTAAAATGTTGACAGTACATAATGGTAGTGGTTTTGCAATAACATCAAAG CCAAGTCCAACTCCGGATCAGGATTCTTATGGAGGAGCTTCTGTGTGTCTTTATTGTAGA GCACATATAGCACACCCTGGCGGAGCAGGAAATTTAGATGGACGCTGTCAATTTAAAGGT TCTTTTGTGCAAATACCTACTACGGAGAAAGATCCTGTTGGATTCTGTCTACGTAACAAG GTTTGCACTGTTTGTCAGTGTTGGATTGGTTATGGATGTCAGTGTGATTCACTTAGACAA CCTAAACCTTCTGTTCAGTCAGTTGCTGTTGCATCTGGTTTTGATAAGAATTATTTAAAC GGGTACGGGGTAGCAGTGAGGCTCGGCTGATACCCCTAGCTAATGGATGTGACCCCGATG TTGTAAAGCGAGCCTTTGATGTTTGTAATAAGGAATCAGCCGGTATGTTTCAAAATTTGA AGCGTAACTGTGCACGATTCCAAGAAGTACGTGATACTGAAGATGGAAATCTTGAGTATT GTGATTCTTATTTTGTGGTTAAACAAACCACTCCTAGTAATTATGAACATGAGAAAGCTT GTTATGAAGACTTAAAGTCAGAAGTAACAGCTGATCATGATTTCTTTGTGTTCAATAAGA ACATTTATAATATTAGTAGGCAGAGGCTTACTAAGTATACTATGATGGATTTTTGCTATG CTTTGCGGCACTTTGACCCAAAGGATTGCGAAGTTCTTAAAGAAATACTTGTCACTTATG GTTGTATAGAAGATTATCACCCTAAGTGGTTTGAAGAGAATAAGGATTGGTACGACCCAA TAGAAAACCCTAAATATTATGCCATGTTGGCTAAAATGGGACCTATTGTACGAGGTGCTT TATTGAATGCTATTGAGTTCGGAAACCTCATGGTTGAAAAAGGTTATGTTGGTGTTATTA CACTTGATAACCAAGATCTTAATGGCAAATTTTATGATTTTGGTGATTTTCAGAAGACAG CGCCTGGTGCTGGTGTTCCTGTTTTTGATACGTATTATTCTTACATGATGCCCATCATAG CCATGACTGATGCGTTGGCACCTGAGAGGTATTTTGAATATGATGTGCATAAGGGTTATA AATCTTATGATCTCCTCAAGTATGATTATACTGAGGAGAAACAAGATTTGTTTCAGAAGT ACTTTAAGTATTGGGATCAAGAGTATCACCCTAACTGTCGCGACTGTAGTGATGACAGGT GTTTGATACATTGTGCAAACTTCAACATCTTGTTTTCTACACTTGTACCGCAGACTTCTT TCGGTAATTTGTGTAGAAAGGTTTTTGTTGATGGTGTACCATTTATAGCTACTTGTGGCT ATCATTCTAAGGAACTTGGTGTTATTATGAATCAAGATAACACCATGTCATTTTCAAAAA TGGGTTTGAGTGAACTCATGGAGTTTGTTGGAGATCGTGGCTTGTTAGTGGGGACATGCA ATAAATTAGTGGATCTTAGAACGTCTTGTTTTAGTGTTTGTGCTTTAGCGTCTGGTATTA CTCATCAAACGGTAAAACCAGGTCACTTTAACAAGGATTTCTACGATTTTGCAGAGAAGG CTGGTATGTTTAAGGAAGGTTCTTCTATACCACTTAAACATTTCTTCTACCCACAGACTG GTAATGCTGCTATAAACGATTATGATTATTATCGTTATAACAGGCCTACCATGTTTGATA TACGTCAACTTTTATTTTGTTTAGAAGTGACTTCTAAATATTTTGAATGTTATGAAGGCG GCTGTATACCAGCAAGCCAAGTTGTAGTTAACAATTTAGATAAGAGTGCAGGTTATCCGT TCAATAAGTTTGGAAAGGCCCGTCTCTATTATGAAATGAGTCTAGAGGAGCAGGACCAAC TCTTTGAGAGTACAAAGAAGAACGTCCTGCCTACTATAACTCAGATGAATTTAAAATATG CCATATCCGCGAAAAATAGAGCGCGTACAGTGGCAGGTGTGTCTATCCTTTCTACTATGA CTAATAGGCAGTTTCATCAGAAGATTCTTAAGTCTATAGTCAACACTAGAAACGCTCCTG TAGTTATTGGAACAACCAAGTTTTATGGCGGTTGGGATAACATGTTGAGAAACCTTATTC AGGGTGTTGAAGACCCGATTCTTATGGGTTGGGATTATCCAAAGTGTGATAGAGCAATGC CTAATTTGTTGCGTATAGCAGCATCTTTAGTACTCGCTCGTAAACACACTAATTGTTGTA CTTGGTCTGAACGCGTTTATAGGTTGTATAATGAATGCGCTCAGGTTTTATCTGAAACTG TCTTAGCTACAGGTGGTATATATGTGAAACCTGGTGGTACTAGCAGTGGAGATGCTACTA CTGCTTATGCAAACAGTGTTTTCAACATAATACAAGCCACATCTGCTAATGTTGCGCGTC TTTTGAGTGTTATAACGCGTGATATTGTATATGATGACATTAAGAGCTTGCAGTATGAAT TGTACCAGCAGGTTTATAGGCGAGTCAATTTTGACCCAGCATTTGTTGAAAAGTTTTATT CTTATTTGTGTAAGAATTTCTCATTGATGATCTTGTCTGACGACGGTGTTGTTTGTTATA ACAACACATTAGCCAAACAAGGTCTTGTAGCAGATATTTCTGGTTTTAGAGAAGTTCTCT ACTATCAGAACAATGTTTTTATGGCTGATTCTAAATGTTGGGTTGAACCAGATTTAGAAA AAGGCCCACATGAATTTTGTTCACAGCACACAATGTTAGTGGAGGTTGATGGTGAGCCTA GATACTTGCCATATCCAGACCCATCACGTATTTTGTGTGCATGTGTTTTTGTAGATGATT TGGATAAGACAGAATCTGTGGCTGTTATGGAGCGTTATATCGCTCTTGCCATAGATGCGT ACCCACTAGTACATCATGAAAATGAGGAGTACAAGAAGGTATTCTTTGTGCTTCTTTCAT ACATCAGAAAACTCTATCAAGAGCTTTCTCAGAATATGCTTATGGACTACTCTTTTGTAA TGGATATAGATAAGGGTAGTAAATTTTGGGAACAGGAGTTCTATGAAAATATGTATAGAG CCCCTACAACATTACAGTGTTGTGGCGTTTGTGTAGTGTGTAATAGTCAAACTATATTGC GCTGTGGTAATTGTATTCGCAAACCATTTTTGTGTTGTAAGTGTTGCTATGACCATGTCA TGCACACAGACCACAAAAATGTTTTGTCTATAAATCCTTACATTTGCTCACAGCCAGGTT GTGGTGAAGCAGATGTTACTAAATTGTACCTCGGAGGTATGTCATACTTCTGCGGTAATC ATAAACCAAAGTTATCAATACCGTTAGTATCTAATGGTACAGTGTTTGGAATTTACAGGG CTAATTGTGCAGGTAGCGAAAATGTTGATGATTTTAATCAACTAGCTACTACTAATTGGT CTACTGTGGAACCTTATATTTTGGCAAATCGTTGTGTAGATTCGTTGAGACGCTTTGCTG CAGAGACAGTAAAAGCTACAGAAGAATTACATAAGCAACAATTTGCTAGTGCAGAAGTGA GAGAAGTACTCTCAGATCGTGAATTGATTCTGTCTTGGGAGCCAGGTAAAACCAGGCCTC CATTGAATAGAAATTATGTTTTCACTGGCTTTCACTTTACTAGAACTAGTAAAGTTCAGC TCGGTGATTTTACATTTGAAAAAGGTGAAGGTAAGGACGTTGTCTATTATCGAGCGACGT CTACTGCTAAATTGTCTGTTGGAGACATTTTTGTTTTAACCTCACACAATGTTGTTTCTC TTATAGCGCCAACGTTGTGTCCTCAGCAAACCTTTTCTAGGTTTGTGAATTTAAGACCTA ATGTGATGGTACCTGCGTGTTTTGTAAATAACATTCCATTGTACCATTTAGTAGGCAAGC AGAAGCGTACTACAGTACAAGGCCCTCCTGGCAGTGGTAAATCCCATTTTGCTATAGGAT TGGCGGCTTACTTTAGTAACGCCCGTGTCGTTTTTACTGCATGCTCTCATGCAGCTGTTG ATGCTTTATGTGAAAAAGCTTTTAAGTTTCTTAAAGTAGATGATTGCACTCGTATAGTAC CTCAAAGGACTACTATCGATTGCTTCTCTAAGTTTAAAGGTAATGACACAGGCAAAAAGT ACATTTTTAGTACTATTAATGCCTTGCCAGAAGTTAGTTGTGACATTCTTTTGGTTGACG AGGTTAGTATGTTGACCAATTACGAATTGTCTTTTATTAATGGTAAGATAAACTATCAAT ATGTTGTGTATGTAGGTGATCCTGCTCAATTACCGGCGCCTCGTACGTTGCTTAACGGTT CACTCTCTCCAAAGGATTATAATGTTGTCACAAACCTTATGGTTTGTGTTAAACCTGACA TTTTCCTTGCAAAGTGTTACCGTTGTCCTAAAGAAATTGTAGATACTGTTTCTACTCTTG TATATGATGGAAAGTTTATTGCAAATAACCCGGAATCACGTCAGTGTTTCAAGGTTATAG TTAATAATGGTAATTCTGATGTAGGACATGAAAGTGGCTCAGCCTACAACATAACTCAAT TAGAATTTGTGAAAGATTTTGTCTGTCGCAATAAGGAATGGCGGGAAGCAACATTCATTT CACCTTATAATGCTATGAACCAGAGAGCCTACCGTATGCTTGGACTTAATGTTCAGACAG TAGACTCGTCTCAAGGTTCGGAGTATGATTATGTTATCTTTTGTGTTACTGCAGATTCGC AGCATGCACTGAATATTAACAGATTCAATGTAGCGCTTACAAGAGCCAAGCGTGGTATAC TAGTTGTCATGCGTCAGCGTGATGAACTATATTCAGCTCTTAAGTTTATAGAGCTTGATA GTGTAGCAAGTCTGCAAGGTACAGGCTTGTTTAAAATTTGCAACAAAGAGTTTAGTGGTG TTCACCCAGCTTATGCAGTCACAACTAAGGCTCTTGCTGCAACTTATAAAGTTAATGATG AACTTGCTGCACTTGTTAACGTGGAAGCTGGTTCAGAAATAACATATAAACATCTTATTT CTTTGTTAGGGTTTAAGATGAGTGTTAATGTTGAAGGCTGCCACAACATGTTTATAACAC GTGATGAGGCTATCCGCAACGTAAGAGGTTGGGTAGGTTTTGATGTAGAAGCAACACATG CTTGCGGTACTAACATTGGTACTAACCTGCCTTTCCAAGTAGGTTTCTCTACTGGTGCAG ACTTTGTAGTTACGCCTGAGGGACTTGTAGATACTTCAATAGGCAATAATTTTGAGCCTG TGAATTCTAAAGCACCTCCAGGTGAACAATTTAATCACTTGAGAGCGTTATTCAAAAGTG CTAAACCTTGGCATGTTGTAAGGCCAAGGATTGTGCAAATGTTAGCGGATAACCTGTGCA ACGTTTCAGATTGTGTAGTGTTTGTCACGTGGTGTCATGGCCTAGAACTAACCACTTTGC GCTATTTTGTTAAAATAGGCAAGGACCAAGTTTGTTCTTGCGGTTCTAGAGCAACAACTT TTAATTCTCATACTCAGGCTTATGCTTGTTGGAAGCATTGCTTGGGTTTTGATTTTGTTT ATAATCCACTCTTAGTGGATATTCAACAGTGGGGTTATTCTGGTAACCTACAATTTAACC ATGATTTGCATTGTAATGTGCATGGACACGCACATGTAGCTTCTGCGGATGCTATTATGA CGCGTTGTCTTGCAATTAATAATGCATTTTGTCAAGATGTCAACTGGGATTTAACTTACC CTCATATAGCAAATGAGGATGAAGTCAATTCTAGCTGTAGATATTTACAACGCATGTATC TTAATGCATGTGTTGATGCTCTTAAAGTTAACGTTGTCTATGATATAGGCAACCCTAAAG GTATAAAATGTGTTAGACGTGGAGACTTAAATTTTAGATTCTATGATAAGAATCCAATAG TACCCAATGTCAAGCAGTTTGAGTATGACTATAATCAGCACAAAGATAAGTTTGCTGATG GTCTTTGTATGTTTTGGAATTGTAATGTGGATTGTTATCCCGACAATTCCTTAGTTTGTA GGTACGACACACGAAATTTGAGTGTGTTTAACCTACCTGGTTGTAATGGTGGTAGCTTGT ATGTTAACAAGCATGCATTCCACACACCTAAATTTGATCGCACTAGCTTTCGTAATTTGA AAGCTATGCCATTCTTTTTCTATGACTCATCGCCTTGCGAGACCATTCAATTGGATGGAG TTGCGCAAGACCTTGTGTCATTAGCTACGAAAGATTGTATCACAAAATGCAACATAGGCG GTGCTGTTTGTAAAAAGCACGCACAAATGTATGCAGATTTTGTGACTTCTTATAATGCAG CTGTTACTGCTGGTTTTACTTTTTGGGTTACTAATAATTTTAACCCATATAATTTGTGGA AAAGTTTTTCAGCTCTCCAGTCTATCGACAATATTGCTTATAATATGTATAAGGGTGGTC ATTATGATGCTATTGCAGGAGAAATGCCCACTATCGTAACTGGAGATAAAGTTTTTGTTA TAGATCAAGGCGTAGAAAAAGCAGTTTTTTTTAATCAAACAATTCTGCCTAGATCTGTAG CGTTTGAGCTGTATGCGAAGAGAAATATTCGCACACTGCCAAACAACCGTATTTTGAAAG GTTTGGGTGTAGATGTGACTAATGGATTTGTAATTTGGGATTACACGAACCAAACACCAC TATACCGTAATACTGTTAAGGTATGTGCATATACAGACATAGAACCAAATGGCCTAATAG TGCTGTATGATGATAGATATGGTGATTACCAGTCTTTTCTAGCTGCTGATAATGCTGTTT TAGTTTCTACACAGTGTTACAAGCGGTATTCGTATGTAGAAATACCGTCAAACCTGCTTG TTCAGAACGGTATTCCGTTAAAAGATGGAGCGAACCTGTATGTTTATAAGCGTGTTAATG GTGCGTTTGTTACGCTACCTAACACATTAAACACACAGGGTCGCAGTTATGAAACTTTTG AACCTCGTAGTGATGTTGAGCGTGATTTTCTCGACATGTCTGAGGAGAGTTTTGTAGAAA AGTATGGTAAAGAATTAGGTCTACAGCACATACTGTATGGTGAAGTTGATAAGCCCCAAT TAGGTGGTTTACACACTGTTATAGGTATGTGCAGACTTTTACGTGCGAATAAGTTGAACG CAAAGTCTGTTACTAATTCTGATTCTGATGTCATGCAAAATTATTTTGTATTGGCAGACA ATGGTTCCTACAAGCAAGTGTGTACTGTTGTGGATTTGCTGCTTGATGATTTCTTAGAAC TTCTTAGGAACATACTGAAAGAGTATGGTACTAATAAGTCTAAAGTTGTAACAGTGTCAA TTGATTACCATAGCATAAATTTTATGACTTGGTTTGAAGATGGCATTATTAAAACATGTT ATCCACAGCTTCAATCAGCATGGACGTGTGGTTATAATATGCCTGAACTTTATAAAGTTC AGAATTGTGTTATGGAACCTTGCAACATTCCTAATTATGGTGTTGGAATAGCGTTGCCAA GTGGTATTATGATGAATGTGGCAAAGTATACACAACTCTGTCAATACCTTTCGAAAACAA CAATGTGTGTACCGCATAATATGCGAGTAATGCATTTTGGAGCTGGAAGTGACAAAGGAG TGGCTCCAGGTAGTACTGTTCTTAAACAATGGCTCCCAGAAGGGACACTCCTTGTCGATA ATGATATTGTAGACTATGTGTCTGATGCACATGTTTCTGTGCTTTCAGATTGCAATAAAT ATAAGACAGAGCACAAGTTTGATCTTGTGATATCTGATATGTATACAGACAATGATTCAA AAAGAAAGCATGAAGGCGTGATAGCCAATAATGGCAATGATGACGTTTTCATATATCTCT CAAGTTTTCTTCGTAATAATTTGGCTCTAGGTGGTAGTTTTGCTGTAAAAGTGACAGAGA CAAGTTGGCACGAAGTTTTATATGACATTGCACAGGATTGTGCATGGTGGACAATGTTTT GTACAGCAGTGAATGCCTCTTCTTCAGAAGCATTCTTGGTTGGTGTTAATTATTTGGGTG CAAGTGAAAAGGTTAAGGTTAGTGGAAAAACGCTGCACGCAAATTATATATTTTGGAGGA ATTGTAATTATTTACAAACCTCTGCTTATAGTATATTTGACGTTGCTAAGTTTGATTTGA GATTGAAAGCAACACCAGTTGTTAATTTGAAAACTGAACAAAAGAGAGACTTAGTGTTTA ATTTAATTAAGTGTGGTAAGTTACTGGTAAGAGATGTTGGTAACACCTCTTTTACTAGTG TACCAAAGTGCCTTTAGACCACCTAATGGTTGGCATTTACACGGGGGTGCTTATGCGGTA GTTAATATTTCTAGCGAATCTAATAATGCAGGCTCTTCACCTGGGTGTATTGTTGGTACT ATTCATGGTGGTCGTGTTGTTAATGCTTCTTCTATAGCTATGACGGCACCGTCATCAGGT ATGGCTTGGTCTAGCAGTCAGTTTTGTACTGCACACTGTAACTTTTCAGATACTACAGTG TTTGTTACACATTGTTATAAATATGATGGGTGTCCTATAACTGGCATGCTTCAAAAGAAT TTTTTACGTGTTTCTGCTATGAAAAATGGCCAGCTTTTCTATAATTTAACAGTTAGTGTA GCTAAGTACCCTACTTTTAAATCATTTCAGTGTGTTAATAATTTAACATCCGTATATTTA AATGGTGATCTTGTTTACACCTCTAATGAGACCACAGATGTTACATCTGCAGGTGTTTAT TTTAAAGCTGGTGGACCTATAACTTATAAAGTTATGAGAGAAGTTAAAGCCCTGGCTTAT TTTGTTAATGGTACTGCACAAGATGTTATTTTGTGTGATGGATCACCTAGAGGCTTGTTA GCATGCCAGTATAATACTGGCAATTTTTCAGATGGCTTTTATCCTTTTATTAATAGTAGT TTAGTTAAGCAGAAGTTTATTGTCTATCGTGAAAATAGTGTTAATACTACTTTTACGTTA CACAATTTCACTTTTCATAATGAGACTGGCGCCAACCCTAATCCTAGTGGTGTTCAGAAT ATTCAAACTTACCAAACACAAACAGCTCAGAGTGGTTATTATAATTTTAATTTTTCCTTT CTGAGTAGTTTTGTTTATAAGGAGTCTAATTTTATGTATGGATCTTATCACCCAAGTTGT AATTTTAGACTAGAAACTATTAATAATGGCTTGTGGTTTAATTCACTTTCAGTTTCAATT GCTTACGGTCCTCTTCAAGGTGGTTGCAAGCAATCTGTCTTTAGTGGTAGAGCAACTTGT TGTTATGCTTATTCATATGGAGGTCCTTCGCTGTGTAAAGGTGTTTATTCAGGTGAGTTA GATCTTAATTTTGAATGTGGACTGTTAGTTTATGTTACTAAGAGCGGTGGCTCTCGTATA CAAACAGCCACTGAACCGCCAGTTATAACTCGACACAATTATAATAATATTACTTTAAAT ACTTGTGTTGATTATAATATATATGGCAGAACTGGCCAAGGTTTTATTACTAATGTAACC GACTCAGCTGTTAGTTATAATTATCTAGCAGACGCAGGTTTGGCTATTTTAGATACATCT GGTTCCATAGACATCTTTGTTGTACAAGGTGAATATGGTCTTACTTATTATTAGGTTAAC CCTTGCGAAGATGTCAACCAGCAGTTTGTAGTTTCTGGTGGTAAATTAGTAGGTATTCTT ACTTCACGTAATGAGACTGGTTCTCAGCTTCTTGAGAACCAGTTTTACATTAAAATCACT AATGGAACACGTCGTTTTAGACGTTCTATTACTGAAAATGTTGGAAATTGCCCTTATGTT AGTTATGGTAAGTTTTGTATAAAACCTGATGGTTCAATTGCCACAATAGTACCAAAACAA TTGGAACAGTTTGTGGCACCTTTACTTAATGTTACTGAAAATGTGCTCATACCTAACAGT TTTAATTTAACTGTTACAGATGAGTACATACAAACGCGTATGGATAAGGTCCAAATTAAT TGTCTGCAGTATGTTTGTGGCAATTCTCTGGATTGTAGAGATTTGTTTCAACAATATGGG CCTGTTTGTGACAACATATTGTCTGTAGTAAATAGTATTGGTCAAAAAGAAGATATGGAA CTTTTGAATTTCTATTCTTCTACTAAACCGGCTGGTTTTAATACACCATTTCTTAGTAAT GTTAGCACTGGTGAGTTTAATATTTCTCTTCTGTTAACAACTCCTAGTAGTCCTAGAAGG CGTTCTTTTATTGAAGACCTTCTATTTACAAGCGTTGAATCTGTTGGATTACCAACAGAT GACGCATACAAAAATTGCACTGCAGGACCTTTAGGTTTTCTTAAGGACCTTGCGTGTGCT CGTGAATATAATGGTTTGCTTGTGTTGCCTCCCATTATAACAGCAGAAATGCAAATTTTG TATACTAGTTCTCTAGTAGCTTCTATGGCTTTTGGTGGTATTACTGCAGCTGGTGCTATA CCTTTTGCCACACAACTGCAGGCTAGAATTAATCACTTGGGTATTACCCAGTCACTTTTG TTGAAGAATCAAGAAAAAATTGCTGCTTCCTTTAATAAGGCCATTGGTCGTATGCAGGAA GGTTTTAGAAGTACATCTCTAGCATTACAACAAATTCAAGATGTTGTTAATAAGCAGAGT GCTATTCTTACTGAGACTATGGCATCACTTAATAAAAATTTTGGTGCTATTTCTTCTATG ATTCAAGAAATCTACCAGCAACTTGACGCCATACAAGCAAATGCTCAAGTGGATCGTCTT ATAACTGGTAGATTGTCATCACTTTCTGTTTTAGCATCTGCTAAGCAGGCGGAGCATATT AGAGTGTCACAACAGCGTGAGTTAGCTACTCAGAAAATTAATGAGTGTGTTAAGTCACAG TCTATTAGGTACTCCTTTTGTGGTAATGGACGACATGTTCTAACCATACCGCAAAATGCA CCTAATGGTATAGTGTTTATACACTTTTCTTATACTCCAGATAGTTTTGTTAATGTTACT GCAATAGTGGGTTTTTGTGTAAAGCCAGCTAATGCTAGTCAGTATGCAATAGTACCCGCT AATGGTAGGGGTATTTTTATACAAGTTAATGGTAGTTACTACATCACAGCACGAGATATG TATATGCCAAGAGCTATTACTGCAGGAGATATAGTTACGCTTACTTCTTGTCAAGCAAAT TATGTAAGTGTAAATAAGACCGTCATTACTACATTCGTAGACAATGATGATTTTGATTTT AATGACGAATTGTCAAAATGGTGGAATGACACTAAGCATGAGCTACCAGACTTTGACAAA TTCAATTACACAGTACCTATACTTGACATTGATAGTGAAATTGATCGTATTCAAGGCGTT ATACAGGGTCTTAATGACTCTTTAATAGACCTTGAAAAACTTTCAATACTCAAAACTTAT ATTAAGTGGCCTTGGTATGTGTGGTTAGCCATAGCTTTTGCCACTATTATCTTCATCTTA ATACTAGGATGGGTTTTCTTCATGACTGGATGTTGTGGTTGTTGTTGTGGATGCTTTGGC ATTATGCCTCTAATGAGTAAGTGTGGTAAGAAATCTTCTTATTACACGACTTTTGATAAC GATGTGGTAACTTAACAATACAGACCTAAAAAGTCTGTTTAATGATTCAAAGTCCCACGT CCTTCCTAATAGTATTAATTTTTCTTTGGTGTAAACTTGTACTAAGTTGTTTTAGAGAGT TTATTATAGCGCTCCAACAACTAATACAAGTTTTACTCCAAATTATCAATAGTAACTTAC AGCCTAGACTGACCCTTTGTCACAGTCTAGACTAATGTTAAACTTAGAAGCAATTATTGA AACTGGTGAGCAAGTGATTCAAAAAATCAGTTTCAATTTACAGCATATTTCAAGTGTATT AAACACAGAAGTATTTGACCCCTTTGACTATTGTTATTACAGAGGAGGTAATTTTTGGGA AATAGAGTCAGCTGAAGATTGTTCAGGTGATGATGAATTTATTGAATAAGTCGCTAGAGG AAAATGGAAGTTTTCTAACAGCGCTTTATATATTTGTAGGATTTTTAGCACTTTATCTTC TAGGTAGAGCACTTCAAGCATTTGTACAGGCTGCTGATGCTTGTTGTTTATTTTGGTATA CATGGGTAGTAATTCCAGGAGCTAAGGGTACAGCCTTTGTATATAAGTATACATATGGTA GAAAACTTAACAATCGGGAATTAGAAGCAGTTATTGTCAACGAGTTTCCTAAGAACGGTT GGAATAATAAAAATCCAGCAAATTTTCAAGATGTCCAACGAGACAAATTGTACTCTTGAC TTTGAACAGTCAGTTGAGCTTTTTAAAGAGTATAATTTATTTATAACTGCATTCTTGTTG TTCTTAACCATAATACTTCAGTATGGCTATGCAACAAGAAGTAAGTTTATTTATATACTG AAAATGATAGTGTTATGGTGCTTTTGGCCCCTTAACATTGCAGTAGGTGTAATTTCATGT ATATACCCACCAAACACAGGAGGTCTTGTCGCAGCGATAATACTTACAGTGTTTGCGTGT CTGTCTTTTGTAGGTTATTGGATCCAGAGTATTAGACTCTTTAAGCGGTGTAGGTCATGG TGGTCATTTAACCCAGAATCTAATGCCGTAGGTTCAATACTCCTAACTAATGGTCAACAA TGTAATTTTGCTATAGAGAGTGTGCCAATGGTGCTTTCTCCAATTATAAAGAATGGTGTT CTTTATTGTGAGGGTCAGTGGCTTGCTAAGTGTGAACCAGACCACTTGCCTAAAGATATA TTTGTTTGTACACCGGATAGACGTAATATCTACCGTATGGTGCAGAAATATACTGGTGAC CAAAGCGGAAATAAGAAACGGTTTGCTACGTTTGTCTATGCAAAGCAGTCAGTAGATACT GGCGAGCTAGAAAGTGTAGCAACAGGAGGGAGTAGTCTTTACACCTAAATGTGTGTGTGT AGAGAGTATTTAAAATTATTCTTTAATAGTGCCTCTATTTTAAGAGCGCATAATAGTATT ATTTTTGAGGATATTAATATAAATCCTCTCTGTTTTATACTCTCTTTTCAAGAGCTATTA TTTAAAAAACAGTTTTTCCACTCTTTTGTGCCAAAAACTATTGTTGTTAATGGTGTAACC TTTCAAGTAGATAATGGAAAAGTCTACTACGAAGGAAAACCAATTTTTCAGAAAGGTTGT TGTAGGTTGTGGTTGAGTTATAAAAAAGATTAAACTACCTACTACACTTATTTTTATAAG AGGCGTTTTATCTTACAAGCGCTTAATAAATACGGACGATGAAATGGCTGACTAGTTTTG TAAGGGCAGTTATTTCATGTTATAAACCCCTATTATTAACTCAATTAAGAGTATTAGATA GGTTAATCTTAGATCATGGACCAAAACACATCTTAACGTGTGTTAGGTGCGTGATTTTGT TTCAATTAGATTTAGTTTATAGGTTGGCGTATACGCCTACTCAATCGCTGGTATGAATAA TAGTAAAGATAATCCTTTTTGCGGAGCAATAGCAAGAAAAGCGCGAATTTATCTGAGAGA AGGATTAGATTGTGTTTACTTTCTTAACAAAGCAGGACAAGCAGAGTCTTGTCCCGCGTG TACCTCTCTAGTATTCCAGGGGAAAACTTGTGAGGAACACAAATATAATAATAATCTTTT GTCATGGCAAGCGGTAAGGCAACTGGAAAGACAGATGCCCCAGCTCCAGTCATCAAACTA GGAGGACCAAAGCCACCTAAAGTTGGTTCTTCTGGAAATGTATCTTGGTTTCAAGCAATA AAAGCCAAGAAGTTAAATTCACCTCCGCCTAAGTTTGAAGGTAGCGGTGTTCCTGATAAT GAAAATCTAAAACCAAGTCAGCAGCATGGATATTGGAGACGCCAAGCTAGGTTTAAGCCA GGTAAAGGTGGAAGAAAACCAGTCCCAGATGCTTGGTATTTTTAGTATACTGGAACAGGA CCAGCCGCTAACCTGAATTGGGGTGATAGCCAAGATGGTATAGTGTGGGTTGCTGGTAAG GGTGCTGATACTAAATTTAGATCTAATCAGGGTACTCGTGACTCTGACAAGTTTGACCAA TATCCGCTACGGTTTTCAGACGGAGGACCTGATGGTAATTTCCGTTGGGATTTCATTCCT CTGAATCGTGGCAGGAGTGGGAGATCAACAGCAGCTTCATCAGCAGCATCTAGTAGAGCA CCATCACGTGAAGTTTCGCGTGGTCGCAGGAGTGGTTCTGAAGATGATCTTATTGCTCGT GCAGCAAGGATAATTCAGGATCAGCAGAAGAAGGGTTCTCGCATTACAAAGGCTAAGGCT GATGAAATGGCTCACCGCCGGTATTGCAAGCGCAGTATTCCACCTAATTATAAGGTTGAT CAAGTGTTTGGTCCCCGTACTAAAGGTAAGGAGGGAAATTTTGGTGATGACAAGATGAAT GAGGAAGGTATTAAGGATGGGCGCGTTACAGCAATGCTCAACCTAGTTCCTAGCAGCCAT GCTTGTCTTTTCGGAAGTAGAGTGACGCCCAGACTTCAACCAGATGGGCTGCACTTGAAA TTTGAATTTACTACTGTGGTCCCACGTGATGATCCGCAGTTTGATAATTATGTAAAAATT TGTGATCAGTGTGTTGATGGTGTAGGAACACGTCCAAAAGATGATGAACCAAGACCAAAG TCACGCTCAAGTTCAAGACCTGCAACAAGAGGAAATTCTCCAGCGCCAAGACAGCAGCGC CCTAAGAAGGAGAAAAAGCCAAAGAAGCAGGATGATGAAGTGGATAAAGCATTGACCTCA GATGAGGAGAGGAACAATGCACAGCTGGAATTTGATGATGAACCCAAGGTAATTAACTGG GGGGATTCAGCGCTAGGAGAGAATGAACTTTGAGTAAAATTGAATAGTAAGAGTTAAGGA AGATAGGCATGTAGCTTGATTACCTACATGTCTATCGCCAGGGAAATGTCTAATTTGTCT ACTTAGTAGCCTGGAAACGAACGGTAGACCCTTAGATTTTAATTTAGTTTAATTTTTAGT TTAGTTTAAGTTAGTTTAGAGTAGGTATAAAGATGCCAGTGGCGGGGCCACGCGGAGTAC GACCGAGGGTACAGCACTAGGACGCCCATTAGGGGAAGAGCTAAATTTTAGTTTAAGTTA AGTTTAATTGGCTATGTATAGTTAAAATTTATAGGCTAGTATAGAGTTAGAGCAAAAAAA AAAAAAAAAAAAAAAAAAAA
In
addition to the structural and accessory genes, two-thirds of a
coronavirus genome comprises the replicase gene (at the 5′ end of
the genome), which is expressed as two polyproteins, pp1a and pp1ab,
in which pp1ab is an extension product of pp1a as a result of a −1
ribosomal shift mechanism. The two polyproteins are cleaved by two
types of virus-encoded proteinases usually resulting in 16
non-structural proteins (Nsp1-16); IBV lacks Nsp1 thereby encoding
Nsp2-16.
Thus
Gene 1 in IBV encodes 15 (16 in other coronaviruses) non-structural
proteins (nsp2-16), which are associated with RNA replication and
transcription.
The
term ‘replicase protein’ is used herein to refer to the pp1a and
pp1ab polyproteins or individual nsp subunits.
The
term ‘replicase gene’ is used herein to refer to a nucleic acid
sequence which encodes for replicase proteins.
colname="1"
colwidth="49pt" align="center"
style="box-sizing: border-box;"colname="2"
colwidth="168pt" align="left" style="box-sizing:
border-box;"TABLE 1 Nsp Protein Key
features colname="1" colwidth="49pt"
align="char" char="." style="box-sizing:
border-box;"colname="2" colwidth="168pt"
align="left" style="box-sizing:
border-box;"1 Conserved within but not between coronavirus
genetic style="box-sizing: border-box;"groups;
potential regulatory functions in the host cell. 2 Dispensable
for MHV and SARS-CoV replication in style="box-sizing:
border-box;"tissue culture 3 Acidic domain; macro
domain with ADRP and poly style="box-sizing:
border-box;"(ADP-ribose)-binding activities; one or two
ZBD- style="box-sizing: border-box;"containing
papain-like proteases; Y domain 4 Transmembrane
domain 5 3C-like main protease, homodimer 6 Transmembrane
domain 7 Interacts with nsp8 to form a hexadecamer
complex 8 Noncannonical RNA polymerase; interacts with
nsp7 to style="box-sizing: border-box;"form a
hexadecameric complex 9 ssRNA-binding protein,
dimer 10 RNA-binding protein, homododecamer,
zinc-binding style="box-sizing: border-box;"domain,
known to interact with nsp14 and nsp16 11 Unknown 12 RNA-dependent
RNA polymerase 13 Zinc-binding domain, NTPase, dNTPase,
5′-to-3′ RNA style="box-sizing: border-box;"and
DNA helicase, RNA 5′-triphosphate 14 3′-to 5′
exoribonuclease, zinc-binding domain and N7- style="box-sizing:
border-box;"methyltransferase 15 Uridylate-specific
endoribonuclease, homohexamer 16 Putative
ribose-2′-O-methyltransferase
The
variant replicase gene encoded by the coronavirus of the present
invention comprises a mutation in one or more of the sections of
sequence encoding nsp-10, nsp-14, nsp-15 or nsp-16.
Nsp10
has RNA-binding activity and appears to be involved in homo and/or
heterotypic interactions within other nsps from the pp1a/pp1ab
region. It adopts an α/β fold comprised of five α-helices, one
310-helix and three β-strands. Two zinc-binding sites have been
identified that are formed by conserved cysteine residues and one
histidine residue (Cys-74/Cys-77/His-83/Cys-90;
Cys-117/Cys-120/Cys-128/Cys-130). The protein has been confirmed to
bind single-stranded and double-stranded RNA and DNA without obvious
specificity. Nsp-10 can be cross-linked with nsp-9, suggesting the
existing of a complex network of protein-protein interactions
involving nsp-7, -8, -9 and -10. In addition, nsp-10 is known to
interact with nsp-14 and nsp-16.
Nsp-14
comprises a 3′-to-5′ exoribonuclease (ExoN) active domain in the
amino-terminal region. SARS-CoV ExoN has been demonstrated to have
metal ion-dependent 3′-to-5′ exoribonuclease activity that acts
on both single-stranded and double-stranded RNA, but not on DNA.
Nsp-14 has been shown to have proof-reading activity. This nsp has
also been shown to have N7-methyltransferase (MT) activity in the
carboxyl-terminal region.
Nsp-15
associated NendoU (nidoviral endoribonuclease, specific for U) RNase
activity has been reported for a number of coronaviruses, including
SARS-CoV, MHV and IBV. The activities were consistently reported to
be significantly enhanced by Mn2+ ions and there was little
activity in the presence of Mg2+ and Ca2+. NendoU cleaves at
the 3′ side of uridylate residues in both single-stranded and
double-stranded RNA. The biologically relevant substrate(s) of
coronavirus NendoUs remains to be identified.
Nsp-16
has been predicted to mediate ribose-2′-O-methyltransferase
(2′-O-MTase) activity and reverse-genetics experiments have shown
that the 2′-O-MTase domain is essential for viral RNA synthesis in
HCoV-229E and SARS-CoV. The enzyme may be involved in the production
of the cap 1 structures of coronavirus RNAs and it may also
cooperate with NendoU and ExoN in other RNA processing pathways.
2′-O-MTase might also methylate specific RNAs to protect them from
NendoU-mediated cleavage.
The
genomic and protein sequences for nsp-10, -14, -15 and -16 are
provided as SEQ ID NO: 2-5 and 6-9, respectively.
colname="1"
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border-box;"colname="2" colwidth="147pt"
align="left" style="box-sizing:
border-box;"(nsp-10 nucleotide sequence- nucleotides 11884-12318 of SEQ ID NO: 1) SEQ ID NO: 2 TCTAAAGGTCATGAGACAGAGGAAGTGGATGCTGTAGGCATTCTCTCACTTTGTTCTTTTGCAGTA GATCCTGCGGATACATATTGTAAATATGTGGCAGCAGGTAATCAACCTTTAGGTAACTGTGTTAAA ATGTTGACAGTACATAATGGTAGTGGTTTTGCAATAACATCAAAGCCAAGTCCAACTCCGGATCAG GATTCTTATGGAGGAGCTTCTGTGTGTCTTTATTGTAGAGCACATATAGCACACCTTGGCGGAGCA GGAAATTTAGATGGACGCTGTCAATTTAAAGGTTCTTTTGTGCAAATACCTACTACGGAGAAAGAT CCTGTTGGATTCTGTCTACGTAACAAGGTTTGCACTGTTTGTCAGTGTTGGATTGGTTATGGATGT CAGTGTGATTCACTTAGACAACCTAAACCTTCTGTTCAG (nsp-14 nucleotide sequence- nucleotides 16938-18500 of SEQ ID NO: 1) SEQ ID NO: 3 GGTACAGGCTTGTTTAAAATTTGCAACAAAGAGTTTAGTGGTGTTCACCCAGCTTATGCAGTCACA ACTAAGGCTCTTGCTGCAACTTATAAAGTTAATGATGAACTTGCTGCACTTGTTAACGTGGAAGCT GGTTCAGAAATAACATATAAACATCTTATTTCTTTGTTAGGGTTTAAGATGAGTGTTAATGTTGAA GGCTGCCACAACATGTTTATAACACGTGATGAGGCTATCCGCAACGTAAGAGGTTGGGTAGGTTTT GATGTAGAAGCAACACATGCTTGCGGTACTAACATTGGTACTAACCTGCCTTTCCAAGTAGGTTTC TCTACTGGTGCAGACTTTGTAGTTACGCCTGAGGGACTTGTAGATACTTCAATAGGCAATAATTTT GAGCCTGTGAATTCTAAAGCACCTCCAGGTGAACAATTTAATCACTTGAGAGCGTTATTCAAAAGT GCTAAACCTTGGCATGTTGTAAGGCCAAGGATTGTGCAAATGTTAGCGGATAACCTGTGCAACGTT TCAGATTGTGTAGTGTTTGTCACGTGGTGTCATGGCCTAGAACTAACCACTTTGCGCTATTTTGTT AAAATAGGCAAGGACCAAGTTTGTTCTTGCGGTTCTAGAGCAACAACTTTTAATTCTCATACTCAG GCTTATGCTTGTTGGAAGCATTGCTTGGGTTTTGATTTTGTTTATAATCCACTCTTAGTGGATATT CAACAGTGGGGTTATTCTGGTAACCTACAATTTAACCATGATTTGCATTGTAATGTGCATGGACAC GCACATGTAGCTTCTGCGGATGCTATTATGACGCGTTGTCTTGCAATTAATAATGCATTTTGTCAA GATGTCAACTGGGATTTAACTTACCCTCATATAGCAAATGAGGATGAAGTCAATTCTAGCTGTAGA TATTTACAACGCATGTATCTTAATGCATGTGTTGATGCTCTTAAAGTTAACGTTGTCTATGATATA GGCAACCCTAAAGGTATTAAATGTGTTAGACGTGGAGACTTAAATTTTAGATTCTATGATAAGAAT CCAATAGTACCCAATGTCAAGCAGTTTGAGTATGACTATAATCAGCACAAAGATAAGTTTGCTGAT GGTCTTTGTATGTTTTGGAATTGTAATGTGGATTGTTATCCCGACAATTCCTTACTTTGTAGGTAC GACACACGAAATTTGAGTGTGTTTAACCTACCTGGTTGTAATGGTGGTAGCTTGTATGTTAACAAG CATGCATTCCACACACCTAAATTTGATCGCACTAGCTTTCGTAATTTGAAAGCTATGCCATTCTTT TTCTATGACTCATCGCCTTGCGAGACCATTCAATTGGATGGAGTTGCGCAAGACCTTGTGTCATTA GCTACGAAAGATTGTATCACAAAATGCAACATAGGCGGTGCTGTTTGTAAAAAGCACGCACAAATG TATGCAGATTTTGTGACTTCTTATAATGCAGCTGTTACTGCTGGTTTTACTTTTTGGGTTACTAAT AATTTTAACCCATATAATTTGTGGAAAAGTTTTTCAGCTCTCCAG (nsp-15 nucleotide sequence- nucleotides 18501-19514 of SEQ ID NO: 1) SEQ ID NO: 4 TCTATCGACAATATTGCTTATAATATGTATAAGGGTGGTCATTATGATGCTATTGCAGGAGAAATG CCCACTATCGTAACTGGAGATAAAGTTTTTGTTATAGATCAAGGCGTAGAAAAAGCAGTTTTTTTT AATCAAACAATTCTGCCTACATCTGTAGCGTTTGAGCTGTATGCGAAGAGAAATATTCGCACACTG CCAAACAACCGTATTTTGAAAGGTTTGGGTGTAGATGTGACTAATGGATTTGTAATTTGGGATTAC ACGAACCAAACACCACTATACCGTAATACTGTTAAGGTATGTGCATATACAGACATAGAACCAAAT GGCCTAATAGTGCTGTATGATGATAGATATGGTGATTACCAGTCTTTTCTAGCTGCTGATAATGCT GTTTTAGTTTCTACACAGTGTTACAAGCGGTATTCGTATGTAGAAATACCGTCAAACCTGCTTGTT CAGAACGGTATTCCGTTAAAAGATGGAGCGAACCTGTATGTTTATAAGCGTGTTAATGGTGCGTTT GTTACGCTACCTAACACAATAAACACACAGGGTCGAAGTTATGAAACTTTTGAACCTCGTAGTGAT GTTGAGCGTGATTTTCTCGACATGTCTGAGGAGAGTTTTGTAGAAAAGTATGGTAAAGAATTAGGT CTACAGCACATACTGTATGGTGAAGTTGATAAGCCCCAATTAGGTGGTTTCCACACTGTTATAGGT ATGTGCAGACTTTTACGTGCGAATAAGTTGAACGCAAAGTCTGTTACTAATTCTGATTCTGATGTC ATGCAAAATTATTTTGTATTGGCAGACAATGGTTCCTACAAGCAAGTGTGTACTGTTGTGGATTTG CTGCTTGATGATTTCTTAGAACTTCTTAGGAACATACTGAAAGAGTATGGTACTAATAAGTCTAAA GTTGTAACAGTGTCAATTGATTACCATAGCATAAATTTTATGACTTGGTTTGAAGATGGCATTATT AAAACATGTTATCCACAGCTTCAA (nsp-16 nucleotide sequence- nucleotides 19515-20423 of SEQ ID NO: 1) SEQ ID NO: 5 TCAGCATGGACGTGTGGTTATAATATGCCTGAACTTTATAAAGTTCAGAATTGTGTTATGGAACCT TGCAACATTCCTAATTATGGTGTTGGAATAGCGTTGCCAAGTGGTATTATGATGAATGTGGCAAAG TATACACAACTCTGTCAATACCTTTCGAAAACAACAATGTGTGTACCGCATAATATGCGAGTAATG CATTTTGGAGCTGGAAGTGACAAAGGAGTGGTGCCAGGTAGTACTGTTCTTAAACAATGGCTCCCA GAAGGGACACTCCTTGTCGATAATGATATTGTAGACTATGTGTCTGATGCACATGTTTCTGTGCTT TCAGATTGCAATAAATATAAGACAGAGCACAAGTTTGATCTTGTGATATCTGATATGTATACAGAC AATGATTCAAAAAGAAAGCATGAAGGCGTGATAGCCAATAATGGCAATGATGACGTTTTCATATAT CTCTCAAGTTTTCTTCGTAATAATTTGGCTCTAGGTGGTAGTTTTGCTGTAAAAGTGACAGAGACA AGTTGGCACGAAGTTTTATATGACATTGCACAGGATTGTGCATGGTGGACAATGTTTTGTACAGCA GTGAATGCCTCTTCTTCAGAAGCATTCTTGATTGGTGTTAATTATTTGGGTGCAAGTGAAAAGGTT AAGGTTAGTGGAAAAACGCTGCACGCAAATTATATATTTTGGAGGAATTGTAATTATTTACAAACC TCTGCTTATAGTATATTTGACGTTGCTAAGTTTGATTTGAGATTGAAAGCAACGCCAGTTGTTAAT TTGAAAACTGAACAAAAGACAGACTTAGTCTTTAATTTAATTAAGTGTGGTAAGTTACTGGTAAGA GATGTTGGTAACACCTCTTTTACTAGTGACTCTTTTGTGTGTACTATGTAG (nsp-10 amino acid sequence) SEQ ID NO: 6 SKGHETEEVDAVGILSLCSFAVDPADTYCKYVAAGNQPLGNCVKMLTVKNGSGFAITSKPSPTPDQ DSYGGASVCLYCRAHIAHPGGAGNLDGRCQFKGSFVQIPTTEKDPVGFCLRNKVCTVCQCWIGYGC QCDSLRQPKPSVQ (nsp-14 amino acid sequence) SEQ ID NO: 7 GTGLFKICNKEFSGVHPAYAVTTKALAATYKVNDELAALVNVEAGSEITYKHLISLLGFKMSVNVE GCHNMFITRDEAIRNVRGWVGFDVEATHACGTNIGTNLPFQVGFSTGADFVVTPEGLVDTSIGNNF EPVNSKAPPGEQFNHLRALFKSAKPWHVVRPRIVQMLADNLCNVSDCVVFVTWCHGLELTTLRYFV KIGKDQVCSCGSRATTFNSHTQAYACWKHCLGFDFVYNPLLVDIQQWGYSGNLQFNHDLHCNVHGH AHVASADAIMTRCLAINNAFCQDVNWDLTYPHIANEDEVNSSCRYLQRMYLNACVDALKVNVVYDI GNPKGIKCVRRGDLNFRFYDKNPIVPNVKQFEYDYNQHKDKFADGLCMFWNCNVDCYPDNSLVCRY DTRNLSVFNLPGCNGGSLYVNKHAFHTPKFDRTSFRNLKAMPFFFYDSSPCETIQLDGVAQDLVSL ATKDCITKCNICGAVCKKKAQMYADFVTSYNAAVTAGFTFWVTNNFNPYNLWKSFSALQ (nsp-15 amino acid sequence) SEQ ID NO: 8 SIDNIAYNMYKGGHYDAIAGEMPTIVTGDKVFVIDQGVEKAVFFNQTILPTSVAFELYAKRNIRTL PNNRILKGLGVDVTNGFVIWDYTNQTPLYRNTVKVCAYTDIEPNGLIVLYDDRYGDYQSFLAADNA VLVSTQCYKRYSYVEIPSNLLVQNGIPLKDGANLYVYKRVNGAFVTLPNTLNTQGRSYETFEPRSD VERDFLDMSEESFVEKYGKELGLQHILYGEVDKPQLGGLHTVIGMCRLLRANKLNAKSVTNSDSDV MQNYFVLADNGSYKQVCTVVDLLLDDFLELLRNILKEYGTNKSKVVTVSIDYHSINFMTWFEDGII KTCYPQLQ (nsp-16 amino acid sequence) SEQ ID NO: 9 SAWTCGYNMPELYKVQNCVMEPCNIPNYGVGIALPSGIMMNVAKYTQLCQYLSKTTMCVPHNMRVM HFGAGSDKGVAPGSTVLKQWLPEGTLLVDNDIVDYVSDAHVSVLSDCNKYKTEHKFDLVISDMYTD NDSKRKHEGVIANNGNDDVFIYLSSFLRNNLALGGSFAVKVTETSWHEVLYDIAQDCAWWTMFCTA VNASSSEAFLVGVNYLGASEKVIWSGKTLHANYIFWRNCNYLQTSAYSIFDVAKFDLRLKATPVVN LKTEQKTDLVFNLIKCGKLLVRDVGNTSFTSDSFVCTM
The
live, attenuated coronavirus of the present invention comprises a
variant replicase gene which causes the virus to have reduced
pathogenicity compared to a coronavirus expressing the corresponding
wild-type gene.
The
term “attenuated” as used herein, refers to a virus that
exhibits said reduced pathogenicity and may be classified as
non-virulent. A live, attenuated virus is a weakened replicating
virus still capable of stimulating an immune response and producing
immunity but not causing the actual illness.
The
term “pathogenicity” is used herein according to its normal
meaning to refer to the potential of the virus to cause disease in a
subject. Typically the pathogenicity of a coronavirus is determined
by assaying disease associated symptoms, for example sneezing,
snicking and reduction in tracheal ciliary activity.
The
term “reduced pathogenicity” is used to describe that the level
of pathogenicity of a coronavirus is decreased, lessened or
diminished compared to a corresponding, wild-type coronavirus.
In
one embodiment, the coronavirus of the present invention has a
reduced pathogenicity compared to the parental M41-CK virus from
which it was derived or a control coronavirus. The control
coronavirus may be a coronavirus with a known pathogenicity, for
example a coronavirus expressing the wild-type replicase protein.
The
pathogenicity of a coronavirus may be assessed utilising methods
well-known in the art. Typically, pathogenicity is assessed by
assaying clinical symptoms in a subject challenged with the virus,
for example a chicken.
As
an illustration, the chicken may be challenged at 8-24 days old by
nasal or ocular inoculation. Clinical symptoms, associated with IBV
infection, may be assessed 3-10 days post-infection. Clinical
symptoms commonly assessed to determine the pathogenicity of a
coronavirus, for example an IBV, include gasping, coughing,
sneezing, snicking, depression, ruffled feathers and loss of
tracheal ciliary activity.
The
variant replicase of the present invention, when expressed in a
coronavirus, may cause a reduced level of clinical symptoms compared
to a coronavirus expressing a wild-type replicase.
For
example a coronavirus expressing the variant replicase may cause a
number of snicks per bird per minute which is less than 90%, less
than 80%, less than 70%, less than 60%, less than 50%, less than
40%, less than 30%, less than 20% or less than 10% of the number of
snicks caused by a virus expressing the wild type replicase.
A
coronavirus expressing a variant replicase according to the present
invention may cause wheezing in less than 70%, less than 60%, less
than 50%, less than 40%, less than 30%, less than 20% or less than
10% of the number of birds in a flock infected with the a virus
expressing the wild type replicase.
A
coronavirus expressing a variant replicase according to the present
invention may result in tracheal ciliary activity which is at least
60%, at least 70%, at least 80%, at least 90% or at least 95% of the
level of tracheal ciliary activity in uninfected birds.
A
coronavirus expressing a variant replicase according to the present
invention may cause clinical symptoms, as defined in Table 2, at a
lower level than a coronavirus expressing the wild type replicase.
colname="1"
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style="box-sizing: border-box;"TABLE 2 IBV severity
limits based on clinical signs:id="CHEM-US-00001"
num="00001" style="box-sizing: border-box;"
The
variant replicase of the present invention, when expressed in a
coronavirus, may cause the virus to replicate at non-pathogenic
levels in ovo.
While
developing vaccines to be administered in ovo to chicken embryos,
attention must be paid to two points: the effect of maternal
antibodies on the vaccines and the effect of the vaccines on the
embryo. Maternal antibodies are known to interfere with active
immunization. For example, vaccines with mild strains do not induce
protective antibody levels when administered to broiler chickens
with maternal antibodies as these strains are neutralized by the
maternal antibody pool.
Thus
a viral particle must be sufficiently efficient at replicating and
propagating to ensure that it is not neutralized by the
maternally-derived antibodies against the virus. Maternally-derived
antibodies are a finite pool of effective antibodies, which decrease
as the chicken ages, and neutralization of the virus in this manner
does not equate to the establishment of long-term immunity for the
embryo/chick. In order to develop long-term immunity against the
virus, the embryo and hatched chicken must develop an appropriate
protective immune response which is distinct to the effect of the
maternally-derived antibodies.
To
be useful for in ovo vaccination, the virus must also not replicate
and propagate at a level which causes it to be pathogenic to the
embryo.
Reduced
pathogenicity in terms of the embryo may mean that the coronavirus
causes less reduction in hatchability compared to a corresponding,
wild-type control coronavirus. Thus the term “without being
pathogenic to the embryo” in the context of the present invention
may mean “without causing reduced hatchability” when compared to
a control coronavirus.
A
suitable variant replicase may be identified using methods which are
known in the art. For example comparative challenge experiments
following in ovo vaccination of embryos with or without
maternally-derived antibodies may be performed (i.e. wherein the
layer has or has not been vaccinated against IBV).
If
the variant replicase enables the virus to propagate at a level
which is too high, the embryo will not hatch or will not be viable
following hatching (i.e. the virus is pathogenic to the embryo). A
virus which is pathogenic to the embryo may kill the embryo.
If
the variant replicase causes a reduction in viral replication and
propagation which is too great, the virus will be neutralised by the
maternally-derived antibodies. Subsequent challenge of the chick
with IBV will therefore result in the development of clinical
symptoms (for example wheezing, snicking, loss of ciliary activity)
and the onset of disease in the challenged chick; as it will have
failed to develop effective immunity against the virus.
As
used herein, the term ‘variant’ is synonymous with ‘mutant’
and refers to a nucleic acid or amino acid sequence which differs in
comparison to the corresponding wild-type sequence.
A
variant/mutant sequence may arise naturally, or may be created
artificially (for example by site-directed mutagenesis). The mutant
may have at least 70, 80, 90, 95, 98 or 99% sequence identity with
the corresponding portion of the wild type sequence. The mutant may
have less than 20, 10, 5, 4, 3, 2 or 1 mutation(s) over the
corresponding portion of the wild-type sequence.
The
term “wild type” is used to mean a gene or protein having a
nucleotide or amino acid sequence which is identical with the native
gene or protein respectively (i.e. the viral gene or protein).
Identity
comparisons can be conducted by eye, or more usually, with the aid
of readily available sequence comparison programs. These
commercially available computer programs can calculate % identity
between two or more sequences. A suitable computer program for
carrying out such an alignment is the GCG Wisconsin Bestfit package
(University of Wisconsin, U.S.A.; Devereux et al., 1984, Nucleic
Acids Research 12:387). Examples of other software that can perform
sequence comparisons include, but are not limited to, the BLAST
package (see Ausubel et al., 1999 ibid—Chapter 18), FASTA (Atschul
et al., 1990, J. Mol. Biol., 403-410) and the GENEWORKS suite of
comparison tools, ClustalX (see Larkin et al. (2007) Clustal W and
Clustal X version 2.0. Bioinformatics, 23:2947-2948). Both BLAST and
FASTA are available for offline and online searching (see Ausubel et
al., 1999 ibid, pages 7-58 to 7-60). However, for some applications,
it is preferred to use the GCG Bestf it program. A new tool, called
BLAST 2 Sequences is also available for comparing protein and
nucleotide sequence (see FEMS Microbiol Lett 1999 174(2): 247-50;
FEMS Microbiol Lett 1999 177(1): 187-8 and
tatiana@ncbi.nlm.nih.gov).
The
sequence may have one or more deletions, insertions or substitutions
of amino acid residues which produce a silent change and result in a
functionally equivalent molecule. Deliberate amino acid
substitutions may be made on the basis of similarity in polarity,
charge, solubility, hydrophobicity, hydrophilicity, and/or the
amphipathic nature of the residues as long as the activity is
retained. For example, negatively charged amino acids include
aspartic acid and glutamic acid; positively charged amino acids
include lysine and arginine; and amino acids with uncharged polar
head groups having similar hydrophilicity values include leucine,
isoleucine, valine, glycine, alanine, asparagine, glutamine, serine,
threonine, phenylalanine, and tyrosine.
Conservative
substitutions may be made, for example according to the Table below.
Amino acids in the same block in the second column and preferably in
the same line in the third column may be substituted for each other:
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align="left" style="box-sizing:
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align="left" style="box-sizing:
border-box;"colname="4" colwidth="49pt"
align="left" style="box-sizing:
border-box;"style="box-sizing:
border-box;"ALIPHATIC Non-polar G A P style="box-sizing:
border-box;"style="box-sizing:
border-box;"style="box-sizing:
border-box;"I L V style="box-sizing:
border-box;"style="box-sizing:
border-box;"Polar- uncharged C S T M style="box-sizing:
border-box;"style="box-sizing:
border-box;"style="box-sizing:
border-box;"N Q style="box-sizing:
border-box;"style="box-sizing:
border-box;"Polar- charged D E style="box-sizing:
border-box;"style="box-sizing:
border-box;"style="box-sizing:
border-box;"K R style="box-sizing:
border-box;"AROMATIC style="box-sizing:
border-box;"H F W Y
The
coronavirus of the present invention may comprise a variant
replicase gene which encodes a protein which comprises a mutation
compared to any one of SEQ ID NO: 6, 7, 8 or 9 which, when expressed
in a coronavirus, causes the virus to have reduced pathogenicity
compared to a coronavirus expressing the corresponding wild-type
replicase.
The
variant replicase gene may encode a protein which comprises at least
one or more amino acid mutations in any combination of nsp-10,
nsp-14, nsp-15 and nsp-16.
The
variant replicase gene of the coronavirus of the present invention
may encode a protein comprising a mutation as defined in the M41 mod
sequences presented in FIG. 10.
The
variant replicase gene of the coronavirus of the present invention
may encode a protein which comprises one or more amino acid
mutations selected from the list of:
The
variant replicase gene of the coronavirus of the present invention
may encode a protein which does not comprise a mutation in nsp-2,
nsp-3, nsp-6 or nsp-13.
The
variant replicase gene of the coronavirus of the present invention
may encode a protein which does not comprise a mutation in nsp10
which corresponds to the threonine to isoleucine mutation caused by
a mutation at nucleotide position 12,008 in the gene reported by
Ammayappan et al. (Arch Virol (2009) 154:495-499).
Ammayappan
et al (as above) reports the identification of sequence changes
responsible for the attenuation of IBV strain Arkansas DPI. The
study identified 17 amino acid changes in a variety of IBV proteins
following multiple passages, approx. 100, of the virus in
embryonated eggs. It was not investigated whether the attenuated
virus (Ark DPI 101) is capable of replicating in the presence of
maternally-derived antibodies against the virus in ovo, without
being pathogenic to the embryo. Given that this virus was produced
by multiple passage in SPF embryonated eggs, similar methodology for
classical IBV vaccines, it is likely that this virus is pathogenic
for embryos. The virus may also be sensitive to maternally-derived
antibodies if the hens were vaccinated with a similar serotype.
The
variant replicase gene of the coronavirus of the present invention
may encode a protein which comprises any combination of one or more
amino acid mutations provided in the list above.
The
variant replicase gene may encode a protein which comprises the
amino acid mutation Pro to Leu at position 85 of SEQ ID NO: 6.
The
variant replicase gene may encode a protein which comprises the
amino acid mutation Val to Leu at position 393 of SEQ ID NO: 7.
The
variant replicase gene may encode a protein which comprises the
amino acid mutation Leu to Ile at position 183 of SEQ ID NO: 8.
The
variant replicase gene may encode a protein which comprises the
amino acid mutation Val to Ile at position 209 of SEQ ID NO: 9.
The
variant replicase gene may encode a protein which comprises the
amino acid mutations Pro to Leu at position 85 of SEQ ID NO: 6, and
Val to Leu at position 393 of SEQ ID NO: 7.
The
variant replicase gene may encode a protein which comprises the
amino acid mutations Pro to Leu at position 85 of SEQ ID NO: 6 Leu
to Ile at position 183 of SEQ ID NO: 8.
The
variant replicase gene may encode a protein which comprises the
amino acid mutations Pro to Leu at position 85 of SEQ ID NO: 6 and
Val to Ile at position 209 of SEQ ID NO: 9.
The
variant replicase gene may encode a protein which comprises the
amino acid mutations Val to Leu at position 393 of SEQ ID NO: 7 and
Leu to Ile at position 183 of SEQ ID NO: 8.
The
variant replicase gene may encode a protein which comprises the
amino acid mutations Val to Leu at position 393 of SEQ ID NO: 7 and
Val to Ile at position 209 of SEQ ID NO: 9.
The
variant replicase gene may encode a protein which comprises the
amino acid mutations Leu to Ile at position 183 of SEQ ID NO: 8 and
Val to Ile at position 209 of SEQ ID NO: 9.
The
variant replicase gene may encode a protein which comprises the
amino acid mutations Pro to Leu at position 85 of SEQ ID NO: 6, Val
to Leu at position 393 of SEQ ID NO: 7 and Leu to Ile at position
183 of SEQ ID NO: 8.
The
variant replicase gene may encode a protein which comprises the
amino acid mutations Pro to Leu at position 85 of SEQ ID NO: 6 Leu
to Ile at position 183 of SEQ ID NO: 8 and Val to Ile at position
209 of SEQ ID NO: 9.
The
variant replicase gene may encode a protein which comprises the
amino acid mutations Pro to Leu at position 85 of SEQ ID NO: 6, Val
to Leu at position 393 of SEQ ID NO: 7 and Val to Ile at position
209 of SEQ ID NO: 9.
The
variant replicase gene may encode a protein which comprises the
amino acid mutations Val to Leu at position 393 of SEQ ID NO: 7, Leu
to Ile at position 183 of SEQ ID NO: 8 and Val to Ile at position
209 of SEQ ID NO: 9.
The
variant replicase gene may encode a protein which comprises the
amino acid mutations Pro to Leu at position 85 of SEQ ID NO: 6, Val
to Leu at position 393 of SEQ ID NO: 7, Leu to Ile at position 183
of SEQ ID NO: 8 and Val to Ile at position 209 of SEQ ID NO: 9.
For
example the nucleotide sequence of the variant replicase gene of the
coronavirus of the present invention may comprise one or more
nucleotide substitutions within the regions selected from the list
of: 11884-12318, 16938-18500, 18501-19514 and 19515-20423 of SEQ ID
NO:1.
For
example the nucleotide sequence of the variant replicase gene of the
coronavirus of the present invention may comprise one or more
nucleotide substitutions selected from the list of:
As
used herein, the term “substitution” is synonymous with the term
mutation and means that the nucleotide at the identified position
differs to that of the wild-type nucleotide sequence.
The
nucleotide sequence may comprise any combination of the nucleotide
substitutions selected from the list of:
The
nucleotide sequence may not comprise a substitution which
corresponds to the C12008T substitution reported by Ammayappan et
al. (as above).
The
nucleotide sequence may be natural, synthetic or recombinant. It may
be double or single stranded, it may be DNA or RNA or combinations
thereof. It may, for example, be cDNA, PCR product, genomic sequence
or mRNA.
A
plasmid is an extra-chromosomal DNA molecule separate from the
chromosomal DNA which is capable of replicating independently of the
chromosomal DNA. They are usually circular and double-stranded.
Plasmids,
or vectors (as they are sometimes known), may be used to express a
protein in a host cell. For example a bacterial host cell may be
transfected with a plasmid capable of encoding a particular protein,
in order to express that protein. The term also includes yeast
artificial chromosomes and bacterial artificial chromosomes which
are capable of accommodating longer portions of DNA.
The
plasmid of the present invention comprises a nucleotide sequence
capable of encoding a defined region of the replicase protein. It
may also comprise one or more additional coronavirus nucleotide
sequence(s), or nucleotide sequence(s) capable of encoding one or
more other coronavirus proteins such as the S gene and/or gene 3.
The
plasmid may also comprise a resistance marker, such as the guanine
xanthine phosphoribosyltransferase gene (gpt) from Escherichia
coli, which confers resistance to mycophenolic acid (MPA) in the
presence of xanthine and hypoxanthine and is controlled by the
vaccinia virus P7.5 early/late promoter.
The
present invention also relates to a recombinant vaccinia virus (rVV)
comprising a variant replicase gene as defined herein.
The
recombinant vaccinia virus (rVV) may be made using a vaccinia-virus
based reverse genetics system.
The
term ‘modified replicase gene’ refers to a replicase gene which
comprises a variant replicase gene as described in connection with
the first aspect of the present invention. Specifically, the term
refers to a gene which is derived from a wild-type replicase gene
but comprises a nucleotide sequence which causes it to encode a
variant replicase protein as defined herein.
The
recombination may involve all or part of the replicase gene. For
example the recombination may involve a nucleotide sequence encoding
for any combination of nsp-10, nsp-14, nsp-15 and/or nsp-16. The
recombination may involve a nucleotide sequence which encodes for an
amino acid mutation or comprises a nucleotide substitution as
defined above.
The
genome of the coronavirus strain may lack the part of the replicase
protein corresponding to the part provided by the plasmid, so that a
modified protein is formed through insertion of the nucleotide
sequence provided by the plasmid.
The
recombining virus is one suitable to allow homologous recombination
between its genome and the plasmid. The vaccinia virus is
particularly suitable as homologous recombination is routinely used
to insert and delete sequences for the vaccinia virus genome.
Methods
for recovering recombinant coronavirus, such as recombinant IBV, are
known in the art (See Britton et al (2005) see page 24; and
PCT/GB2010/001293).
For
example, the DNA from the recombining virus from step (iv) may be
inserted into a plasmid and used to transfect cells which express
cytoplasmic T7 RNA polymerase. The cells may, for example be
pre-infected with a fowlpox virus expressing T7 RNA polymerase.
Recombinant coronavirus may then be isolated, for example, from the
growth medium.
When
the plasmid is inserted into the vaccinia virus genome, an unstable
intermediate is formed. Recombinants comprising the plasmid may be
selected for e.g. using a resistance marker on the plasmid.
Positive
recombinants may then be verified to contain the modified replicase
gene by, for example, PCR and sequencing.
Large
stocks of the recombining virus including the modified replicase
gene (e.g. recombinant vaccinia virus, (rVV) may be grown up and the
DNA extracted in order to carry out step (v)).
Suitable
reverse genetics systems are known in the art (Casais et al (2001)
J. Virol 75:12359-12369; Casais et al (2003) J. Virol. 77:9084-9089;
Britton et al (2005) J. Virological Methods 123:203-211; Armesto et
al (2008) Methods in Molecular Biology 454:255-273).
Coronavirus
particles may be harvested, for example from the supernatant, by
methods known in the art, and optionally purified.
Thus
the present invention also provides a method for producing a
coronavirus which comprises the following steps:
The
present invention also provides a cell capable of producing a
coronavirus according to the invention using a reverse genetics
system. For example, the cell may comprise a recombining virus
genome comprising a nucleotide sequence capable of encoding the
replicase gene of the present invention.
The
cell may be able to produce recombinant recombining virus (e.g.
vaccinia virus) containing the replicase gene.
Alternatively
the cell may be capable of producing recombinant coronavirus by a
reverse genetics system. The cell may express or be induced to
express T7 polymerase in order to rescue the recombinant viral
particle.
The
coronavirus may be used to produce a vaccine. The vaccine may by a
live attenuated form of the coronavirus of the present invention and
may further comprise a pharmaceutically acceptable carrier. As
defined herein, “pharmaceutically acceptable carriers” suitable
for use in the invention are well known to those of skill in the
art. Such carriers include, without limitation, water, saline,
buffered saline, phosphate buffer, alcohol/aqueous solutions,
emulsions or suspensions. Other conventionally employed diluents and
excipients may be added in accordance with conventional techniques.
Such carriers can include ethanol, polyols, and suitable mixtures
thereof, vegetable oils, and injectable organic esters. Buffers and
pH adjusting agents may also be employed. Buffers include, without
limitation, salts prepared from an organic acid or base.
Representative buffers include, without limitation, organic acid
salts, such as salts of citric acid, e.g., citrates, ascorbic acid,
gluconic acid, histidine-Hel, carbonic acid, tartaric acid, succinic
acid, acetic acid, or phthalic acid, Iris, trimethanmine
hydrochloride, or phosphate buffers. Parenteral carriers can include
sodium chloride solution, Ringer's dextrose, dextrose, trehalose,
sucrose, and sodium chloride, lactated Ringer's or fixed oils.
Intravenous carriers can include fluid and nutrient replenishers,
electrolyte replenishers, such as those based on Ringer's dextrose
and the like. Preservatives and other additives such as, for
example, antimicrobials, antioxidants, chelating agents (e.g.,
EDTA), inert gases and the like may also be provided in the
pharmaceutical carriers. The present invention is not limited by the
selection of the carrier. The preparation of these pharmaceutically
acceptable compositions, from the above-described components, having
appropriate pH isotonicity, stability and other conventional
characteristics is within the skill of the art. See, e.g., texts
such as Remington: The Science and Practice of Pharmacy, 20th ed,
Lippincott Williams & Wilkins, pub!., 2000; and The Handbook of
Pharmaceutical Excipients, 4.sup.th edit., eds. R. C. Rowe et al,
APhA Publications, 2003.
The
vaccine of the invention will be administered in a “therapeutically
effective amount”, which refers to an amount of an active
ingredient, e.g., an agent according to the invention, sufficient to
effect beneficial or desired results when administered to a subject
or patient. An effective amount can be administered in one or more
administrations, applications or dosages. A therapeutically
effective amount of a composition according to the invention may be
readily determined by one of ordinary skill in the art. In the
context of this invention, a “therapeutically effective amount”
is one that produces an objectively measured change in one or more
parameters associated Infectious Bronchitis condition sufficient to
effect beneficial or desired results. An effective amount can be
administered in one or more administrations. For purposes of this
invention, an effective amount of drug, compound, or pharmaceutical
composition is an amount sufficient to reduce the incidence of
Infectious Bronchitis. As used herein, the term “therapeutic”
encompasses the full spectrum of treatments for a disease, condition
or disorder. A “therapeutic” agent of the invention may act in a
manner that is prophylactic or preventive, including those that
incorporate procedures designed to target animals that can be
identified as being at risk (pharmacogenetics); or in a manner that
is ameliorative or curative in nature; or may act to slow the rate
or extent of the progression of at least one symptom of a disease or
disorder being treated.
The
present invention also relates to a method for producing such a
vaccine which comprises the step of infecting cells, for example
Vero cells, with a viral particle comprising a replicase protein as
defined in connection with the first aspect of the invention.
To
“treat” means to administer the vaccine to a subject having an
existing disease in order to lessen, reduce or improve at least one
symptom associated with the disease and/or to slow down, reduce or
block the progression of the disease.
To
“prevent” means to administer the vaccine to a subject who has
not yet contracted the disease and/or who is not showing any
symptoms of the disease to prevent or impair the cause of the
disease (e.g. infection) or to reduce or prevent development of at
least one symptom associated with the disease.
The
disease may be any disease caused by a coronavirus, such as a
respiratory disease and and/or gastroenteritis in humans and
hepatitis, gastroenteritis, encephalitis, or a respiratory disease
in other animals.
The
disease may be infectious bronchitis (IB); Porcine epidemic
diarrhoea; Transmissible gastroenteritis; Mouse hepatitis virus;
Porcine haemagglutinating encephalomyelitis; Severe acute
respiratory syndrome (SARS); or Bluecomb disease.
The
vaccine may be administered to hatched chicks or chickens, for
example by eye drop or intranasal administration. Although accurate,
these methods can be expensive e.g. for large broiler flocks.
Alternatives include spray inoculation of administration to drinking
water but it can be difficult to ensure uniform vaccine application
using such methods.
The
vaccine may be provided in a form suitable for its administration,
such as an eye-dropper for intra-ocular use.
The
vaccine may be administered by in ovo inoculation, for example by
injection of embryonated eggs. In ovo vaccination has the advantage
that it provides an early stage resistance to the disease. It also
facilitates the administration of a uniform dose per subject, unlike
spray inoculation and administration via drinking water.
The
vaccine may be administered to any suitable compartment of the egg,
including allantoic fluid, yolk sac, amnion, air cell or embryo. It
may be administered below the shell (aircell) membrane and
chorioallantoic membrane.
Usually
the vaccine is injected into embryonated eggs during late stages of
embryonic development, generally during the final quarter of the
incubation period, such as 3-4 days prior to hatch. In chickens, the
vaccine may be administered between day 15-19 of the 21-day
incubation period, for example at day 17 or 18.
The
process can be automated using a robotic injection process, such as
those described in WO 2004/078203.
The
vaccine may be administered together with one or more other
vaccines, for example, vaccines for other diseases, such as
Newcastle disease virus (NDV). The present invention also provides a
vaccine composition comprising a vaccine according to the invention
together with one or more other vaccine(s). The present invention
also provides a kit comprising a vaccine according to the invention
together with one or more other vaccine(s) for separate, sequential
or simultaneous administration.
The
vaccine or vaccine composition of the invention may be used to treat
a human, animal or avian subject. For example, the subject may be a
chick, chicken or mouse (such as a laboratory mouse, e.g. transgenic
mouse).
Typically,
a physician or veterinarian will determine the actual dosage which
will be most suitable for an individual subject or group of subjects
and it will vary with the age, weight and response of the particular
subject(s).
The
composition may optionally comprise a pharmaceutically acceptable
carrier, diluent, excipient or adjuvant. The choice of
pharmaceutical carrier, excipient or diluent can be selected with
regard to the intended route of administration and standard
pharmaceutical practice. The pharmaceutical compositions may
comprise as (or in addition to) the carrier, excipient or diluent,
any suitable binder(s), lubricant(s), suspending agent(s), coating
agent(s), solubilising agent(s), and other carrier agents that may
aid or increase the delivery or immunogenicity of the virus.
The
invention will now be further described by way of Examples, which
are meant to serve to assist one of ordinary skill in the art in
carrying out the invention and are not intended in any way to limit
the scope of the invention.
EXAMPLESExample
1—Generation of an IBV Reverse Genetics System Based on M41-CK
A
M41-CK full-length cDNA was produced by replacement of the Beaudette
cDNA in the Vaccinia virus reverse genetics system previously
described in PCT/GB2010/001293 (herein incorporated by reference)
with synthetic cDNA derived from the M41 consensus sequence.
The
IBV cDNA within recombinant Vaccinia virus (rVV) rVV-BeauR-Rep-M41
structure described in Armesto, Cavanagh and Britton (2009). PLoS
ONE 4(10): e7384. doi:10.1371/journal.pone.0007384, which consisted
of the replicase derived from IBV Beaudette strain and the
structural and accessory genes and 3′ UTR from IBV M41-CK, was
further modified by replacement of the Beaudette 5′ UTR-Nsp2-Nsp3
sequence with the corresponding sequence from IBV M41-CK. The
resulting IBV cDNA consisted of 5′ UTR-Nsp2-Nsp3 from M41,
Nsp4-Nsp16 from Beaudette and the structural and accessory genes and
3′ UTR from M41. This cDNA was further modified by the deletion of
the Beaudette Nsp4-Nsp16 sequence. The resulting cDNA, lacking
Nsp4-16, was modified in four further steps in which the deleted
Nsps were sequentially replaced with the corresponding sequences
from M41-CK, the replacement cDNAs represented M41-CK Nsp4-8,
Nsp9-12, Nsp12-14 and finally Nsp15-16. Each replacement cDNA
contained approx. 500 nucleotides at the 5′ end corresponding to
the 3′ most M41 sequence previously inserted and approx. 500
nucleotides at the 3′ end corresponding to the M41 S gene
sequence. This allowed insertion of the M41 cDNA sequence by
homologous recombination and sequential addition of contiguous M41
replicase gene sequence. The synthetic cDNAs containing the
M41-derived Nsp sequences were added by homologous recombination
utilising the inventor's previous described transient dominant
selection (IDS) system (see PCT/GB2010/001293). The M41-derived
cDNAs containing sequence corresponding to the M41 Nsps-10, -14, -15
and -16 contained the modified amino acids at positions 85, 393, 183
and 209, respectively, as indicated in FIG. 10.
A
full-length cDNA representing the genome of M41-CK was generated in
Vaccinia virus representing the synthetic sequences. Two rIBVs,
M41-R-6 and M41-R-12, were rescued and shown to grow in a similar
manner as M41-CK (FIG. 1).
Example
2—Determining the Pathogenicity of Rescued M41 Viruses
The
viruses rescued in Example 1 were used to infect 8-day-old specific
pathogen free (SPF) chicks by ocular and nasal inoculation to test
them for pathogenicity, as observed by clinical signs on a daily
basis 3-7 days post-infection and for ciliary activity days 4 and 6
post-infection. Loss of ciliary activity is a well-established
method for determining the pathogenicity of IBV. The two M41-R
viruses were found to be apathogenic when compared to M41-CK though
they did show some clinical signs in comparison to uninfected
control chicks (FIG. 2) and some but inconsistent loss in ciliary
activity (FIG. 3).
Thus,
the M41-R molecular clones of M41-CK were not pathogenic when
compared to the parental virus M41-CK.
The
inventors identified several nucleotide differences in the M41-R
compared to the M41-CK sequences. The majority of these were
synonymous mutations, as the nucleotide change did not affect the
amino acid sequence of the protein associated with the sequence.
However, four non-synonymous mutations were identified in the IBV
replicase gene specific to Nsp-10, Nsp-14, Nsp-15 and Nsp-16
components of the replicase gene, these mutations resulted in amino
acid changes (Table 3).
colname="1"
colwidth="217pt" align="center"
style="box-sizing: border-box;"TABLE 3 Non-Synonymous
mutations identified in the Nsps of M41-R full-length
genome colname="1" colwidth="35pt"
align="center" style="box-sizing:
border-box;"colname="2" colwidth="56pt"
align="center" style="box-sizing:
border-box;"colname="3" colwidth="42pt"
align="center" style="box-sizing:
border-box;"colname="4" colwidth="84pt"
align="center" style="box-sizing: border-box;"Region
of Nucleotide Nucleotide Replicase position Mutation Amino
Acid
Change Nsp10 12137 C→T Pro→Leu Nsp14 18114 G→C Val→Leu Nsp15 19047 T→A Leu→Ile Nsp16 20139 G→A Val→Ile
Example
3—Repair of M41-R rIBVs
In
order to determine whether the identified mutations were responsible
for the loss of pathogenicity associated with M41-R, the Nsp10
mutation was repaired and the mutations in Nsp-14, -15 & -16
were repaired and shown to grow in a similar manner as M41-CK (FIG.
9). The inventors thus generated the rIBVs, M41R-nsp10rep and
M41R-nsp14, 15, 16rep, using synthetic cDNAs containing the correct
nucleotides utilising the inventor's previous described (TDS) system
(see PCT/GB2010/001293).
The
rIBVs were assessed for pathogenicity in chicks as described
previously. Both rIBVs showed increased pathogenicity when compared
to M41-R but not to the level observed with M41-CK (FIGS. 4 and 5).
M41R-nsp14, 15, 16rep gave more clinical signs and more reduction in
ciliary activity than M41R-nsp10rep, overall these results indicated
that the changes associated with the four Nsps appear to affect
pathogenicity.
To
determine the roles of the Nsps in pathogenicity the full-length
cDNA corresponding to M41R-nsp10rep was used to repair the mutations
in Nsps14, 15 & 16 using a synthetic cDNA containing the correct
nucleotides utilising the TDS system.
The
rIBVs were shown to grow in a similar manner as M41-CK (FIG. 9) and
assessed for pathogenicity as described previously. M41-K (in which
all four mutations had been repaired) resulted in clinical signs and
100% loss of ciliary activity (complete ciliostasis) by 4 days
post-infection (FIGS. 6, 7 & 8). The other rIBVs demonstrated
varying levels of pathogenicity, apart from M41R-nsp10, 15, 16rep,
which was essentially apathogenic. These results confirmed that
repair of all four Nsps restored pathogenicity to M41-R; again
supporting the previous evidence that the mutations described in the
four Nsps are implicated in attenuating M41-CK.
The
inventors also generated rIBV M41R-nsp 10, 14 rep (nsp 10 and 14 are
repaired, nsp 15 and 16 contain mutations) and rIBV M41R-nsp 10, 16
rep (nsp 10 and 16 are repaired, nsp 14 and 15 contain mutations)
and assessed the pathogenicity of these viruses.
rIBV
M41R-nsp 10, 14 rep less pathogenic than M41-K but caused around 50%
ciliostasis on days 4-6 post-infection. rIBV M41R-nsp 10, 16 rep was
almost apathogenic and caused no ciliostasis (see FIG.
11style="box-sizing: border-box;"a-c).
Thus
the genome associated with M41-R is a potential backbone genome for
a rationally attenuated IBV.
Example
4—Vaccination/Challenge Study with M41-R
Candidate
vaccine viruses were tested in studies in which fertilized chicken
eggs were vaccinated in ovo at 18 days embryonation and in which the
hatchability of the inoculated eggs was determined. The clinical
health of the chickens was investigated and the chickens were
challenged at 21 days of age with a virulent IB M41 challenge virus
at 103.65 EID50 per dose.
Clinical
signs were investigated after challenge protection by the vaccine
and a ciliostasis test was performed at 5 days after challenge to
investigate the effect of the challenge viruses on movement of the
cilia and protection by the vaccine against ciliostasis (inhibition
of cilia movement).
The
design of the experiment is given in Table 4 and the clinical
results are given in Table 5. Hatchability of the eggs inoculated
with IB M41-R was good and chickens were healthy. IB M41-R protected
against clinical signs after challenge in the broilers (placebo:
19/19 affected, 1B M41-R: 3/18 affected and 1 dead). The results of
the ciliostasis test are given in Table 6. IB M41-R generated
protection against ciliostasis.
colname="1"
colwidth="273pt" align="center"
style="box-sizing: border-box;"TABLE 4 Design of a
hatchability, safety, efficacy study in commercial eggs colname="1"
colwidth="35pt" align="left" style="box-sizing:
border-box;"colname="2" colwidth="42pt"
align="left" style="box-sizing:
border-box;"colname="3" colwidth="28pt"
align="left" style="box-sizing:
border-box;"colname="4" colwidth="28pt"
align="left" style="box-sizing:
border-box;"colname="5" colwidth="35pt"
align="left" style="box-sizing:
border-box;"colname="6" colwidth="42pt"
align="left" style="box-sizing:
border-box;"colname="7" colwidth="28pt"
align="left" style="box-sizing:
border-box;"colname="8" colwidth="35pt"
align="center" style="box-sizing:
border-box;"style="box-sizing:
border-box;"style="box-sizing:
border-box;"EIDstyle="box-sizing: border-box; font-size:
12px; line-height: 0; position: relative; vertical-align: baseline;
bottom: -0.25em;"501 Route Day(s) Day(s) End Nr.
of style="box-sizing:
border-box;"Treatment per of of of of eggs
per Treatment Description dose Admin Admin Challengestyle="box-sizing:
border-box; font-size: 12px; line-height: 0; position: relative;
vertical-align: baseline; top:
-0.5em;"2 Study treatment T01 None NA NA NA NA NA 30 T02 IB
M41-R 10style="box-sizing: border-box; font-size: 12px;
line-height: 0; position: relative; vertical-align: baseline; top:
-0.5em;"4 In ovo 18 days At 21 days At
26 30 NTX Saline NA In ovo embryo- of
age, 20 days 30 style="box-sizing:
border-box;"style="box-sizing:
border-box;"style="box-sizing:
border-box;"style="box-sizing:
border-box;"nation chickens of age style="box-sizing:
border-box;"style="box-sizing:
border-box;"style="box-sizing:
border-box;"style="box-sizing:
border-box;"style="box-sizing: border-box;"per
group style="box-sizing: border-box; font-size: 12px;
line-height: 0; position: relative; vertical-align: baseline; top:
-0.5em;"1Dose volume 0.1 ml, NA, not
applicable. style="box-sizing: border-box; font-size:
12px; line-height: 0; position: relative; vertical-align: baseline;
top: -0.5em;"2103.65 EID50 per dose.
colname="1"
colwidth="273pt" align="center"
style="box-sizing: border-box;"TABLE 5 Hatch
percentages and clinical data before and after challenge in
commercial chickens, for design see Table 1. colname="1"
colwidth="42pt" align="left" style="box-sizing:
border-box;"colname="2" colwidth="35pt"
align="center" style="box-sizing:
border-box;"colname="3" colwidth="28pt"
align="center" style="box-sizing:
border-box;"colname="4" colwidth="77pt"
align="center" style="box-sizing:
border-box;"colname="5" colwidth="91pt"
align="center" style="box-sizing:
border-box;"style="box-sizing:
border-box;"style="box-sizing:
border-box;"style="box-sizing:
border-box;"Before After style="box-sizing:
border-box;"style="box-sizing:
border-box;"style="box-sizing:
border-box;"challenge challenge colname="1"
colwidth="42pt" align="left" style="box-sizing:
border-box;"colname="2" colwidth="35pt"
align="center" style="box-sizing:
border-box;"colname="3" colwidth="28pt"
align="center" style="box-sizing:
border-box;"colname="4" colwidth="35pt"
align="center" style="box-sizing:
border-box;"colname="5" colwidth="42pt"
align="center" style="box-sizing:
border-box;"colname="6" colwidth="35pt"
align="center" style="box-sizing:
border-box;"colname="7" colwidth="56pt"
align="left" style="box-sizing:
border-box;"style="box-sizing:
border-box;"Hatch/ Vital/ Deaths/ Symptoms/ Deaths/ Symptoms/ Treatment total total total total total total colname="1"
colwidth="42pt" align="left" style="box-sizing:
border-box;"colname="2" colwidth="35pt"
align="center" style="box-sizing:
border-box;"colname="3" colwidth="196pt"
align="center" style="box-sizing:
border-box;"None 28/30 Euthanized directly after
hatch for blood collection colname="1"
colwidth="42pt" align="left" style="box-sizing:
border-box;"colname="2" colwidth="35pt"
align="center" style="box-sizing:
border-box;"colname="3" colwidth="28pt"
align="center" style="box-sizing:
border-box;"colname="4" colwidth="35pt"
align="center" style="box-sizing:
border-box;"colname="5" colwidth="42pt"
align="center" style="box-sizing:
border-box;"colname="6" colwidth="35pt"
align="center" style="box-sizing:
border-box;"colname="7" colwidth="56pt"
align="left" style="box-sizing: border-box;"IB
M41-R 28/30 28/28 1/20 0/19 1/19 3/18style="box-sizing:
border-box; font-size: 12px; line-height: 0; position: relative;
vertical-align: baseline; top: -0.5em;"1,
7 Saline 29/30 29/29 1/20 0/19 0/19 19/19style="box-sizing:
border-box; font-size: 12px; line-height: 0; position: relative;
vertical-align: baseline; top: -0.5em;"1, 2, 3, 4, 5, 6,
7 style="box-sizing: border-box; font-size: 12px;
line-height: 0; position: relative; vertical-align: baseline; top:
-0.5em;"1Disturbed respiratory system style="box-sizing:
border-box; font-size: 12px; line-height: 0; position: relative;
vertical-align: baseline; top: -0.5em;"2Whizzing style="box-sizing:
border-box; font-size: 12px; line-height: 0; position: relative;
vertical-align: baseline; top: -0.5em;"3Change of
voice style="box-sizing: border-box; font-size: 12px;
line-height: 0; position: relative; vertical-align: baseline; top:
-0.5em;"4Breathing difficult style="box-sizing:
border-box; font-size: 12px; line-height: 0; position: relative;
vertical-align: baseline; top: -0.5em;"5Swollen intra-orbital
sinuses style="box-sizing: border-box; font-size: 12px;
line-height: 0; position: relative; vertical-align: baseline; top:
-0.5em;"6Uneven growth style="box-sizing: border-box;
font-size: 12px; line-height: 0; position: relative; vertical-align:
baseline; top: -0.5em;"7Weak
colname="1"
colwidth="217pt" align="center"
style="box-sizing: border-box;"TABLE 6 Results of the
ciliostasis test after challenge, for design see Table
1. colname="1" colwidth="56pt"
align="center" style="box-sizing:
border-box;"colname="2" colwidth="63pt"
align="center" style="box-sizing:
border-box;"colname="3" colwidth="98pt"
align="center" style="box-sizing:
border-box;"Treatment Protected/total Percentage
protection Saline 0/19 0% IB M41R 5/18 28%
The
design of the study in SPF eggs is given in Table 7 and is similar
with the design of the studies with commercial broilers, but the
vaccination dose for 1B M41-R was higher, (105 EID50 per
dose).
The
results (Table 8) show that the hatch percentage for IB M41-R hatch
was low, and 19 of 40 hatched and the chicks were weak. Eight chicks
died. The remaining 11 chickens were challenged and 11 of the chicks
hatched from the eggs which had been inoculated with saline were
challenged.
In
the ciliostasis test after challenge it appeared that all chickens
vaccinated in ovo with IB M41-R were protected, whereas none of the
controls was protected, see Table 9.
colname="1"
colwidth="273pt" align="center"
style="box-sizing: border-box;"TABLE 7 Design of a
hatchability, safety, efficacy study in SPF eggs colname="1"
colwidth="35pt" align="left" style="box-sizing:
border-box;"colname="2" colwidth="42pt"
align="left" style="box-sizing:
border-box;"colname="3" colwidth="28pt"
align="left" style="box-sizing:
border-box;"colname="4" colwidth="28pt"
align="left" style="box-sizing:
border-box;"colname="5" colwidth="35pt"
align="left" style="box-sizing:
border-box;"colname="6" colwidth="42pt"
align="left" style="box-sizing:
border-box;"colname="7" colwidth="28pt"
align="left" style="box-sizing:
border-box;"colname="8" colwidth="35pt"
align="center" style="box-sizing:
border-box;"style="box-sizing:
border-box;"style="box-sizing:
border-box;"EIDstyle="box-sizing: border-box; font-size:
12px; line-height: 0; position: relative; vertical-align: baseline;
bottom: -0.25em;"501 Route Day Day End Nr.
of style="box-sizing:
border-box;"Treatment per of of of of eggs
per Treatment Description dose Admin Admin Challengestyle="box-sizing:
border-box; font-size: 12px; line-height: 0; position: relative;
vertical-align: baseline; top: -0.5em;"2 Study treatment T01 IB
M41-R 10style="box-sizing: border-box; font-size: 12px;
line-height: 0; position: relative; vertical-align: baseline; top:
-0.5em;"5 In ovo 18 days At 21 days At
26 40 style="box-sizing:
border-box;"style="box-sizing:
border-box;"style="box-sizing:
border-box;"style="box-sizing: border-box;"embryo- of
age days T04 Saline NA In
ovo nation style="box-sizing: border-box;"of
age 40 NTX NA NA NA style="box-sizing:
border-box;"NA style="box-sizing:
border-box;"10 style="box-sizing: border-box;
font-size: 12px; line-height: 0; position: relative; vertical-align:
baseline; top: -0.5em;"1Dose volume 0.1 ml, NA, not
applicable. style="box-sizing: border-box; font-size:
12px; line-height: 0; position: relative; vertical-align: baseline;
top: -0.5em;"2Challenge dose 103.3 EID50 in 0.2 ml.
colname="1"
colwidth="266pt" align="center"
style="box-sizing: border-box;"TABLE 8 Hatch
percentages and clinical data before and after challenge in SPF
chickens, for design see Table 7. colname="1"
colwidth="42pt" align="left" style="box-sizing:
border-box;"colname="2" colwidth="35pt"
align="center" style="box-sizing:
border-box;"colname="3" colwidth="28pt"
align="center" style="box-sizing:
border-box;"colname="4" colwidth="77pt"
align="center" style="box-sizing:
border-box;"colname="5" colwidth="84pt"
align="center" style="box-sizing:
border-box;"style="box-sizing:
border-box;"style="box-sizing:
border-box;"style="box-sizing:
border-box;"Before After style="box-sizing:
border-box;"style="box-sizing:
border-box;"style="box-sizing:
border-box;"challenge challenge colname="1"
colwidth="42pt" align="left" style="box-sizing:
border-box;"colname="2" colwidth="35pt"
align="center" style="box-sizing:
border-box;"colname="3" colwidth="28pt"
align="center" style="box-sizing:
border-box;"colname="4" colwidth="35pt"
align="center" style="box-sizing:
border-box;"colname="5" colwidth="42pt"
align="center" style="box-sizing:
border-box;"colname="6" colwidth="35pt"
align="center" style="box-sizing:
border-box;"colname="7" colwidth="49pt"
align="center" style="box-sizing:
border-box;"style="box-sizing:
border-box;"Hatch/ Vital/ Deaths/ Symptoms/ Deaths/ Symptoms/ Treatment total total total total total total IB
M41-R 19/40 11/40 8/40 weak 0 0 Saline 30/40 30/40 0 — 0 0 NA 9/10 9/10 0 — — —
colname="1"
colwidth="217pt" align="center"
style="box-sizing: border-box;"TABLE 9 Results of the
ciliostasis test after challenge, for design see Table
7. colname="1" colwidth="56pt"
align="center" style="box-sizing:
border-box;"colname="2" colwidth="63pt"
align="center" style="box-sizing:
border-box;"colname="3" colwidth="98pt"
align="center" style="box-sizing:
border-box;"Treatment Protected/total Percentage
protection Saline 0/11 0% IB
M41R 11/11 100%
In
conclusion, IB M41-R was safe in commercial eggs, generated
protection against clinical signs and to an extent against
ciliostasis.
In
SPF eggs vaccinated with IB M41 R a relatively low number of
chickens hatched. This may be due to the 105 EID50 per egg
of 1B M41-R used. This was 10-fold higher than the dose used in
earlier studies in which there was a higher level of hatchability.
The lower hatch percentages may also be caused by a particularly
high susceptibility of the batch of SPF eggs for viruses, as in
other studies the level of embryo mortality was also higher that had
previously been observed.
After
challenge all surviving chickens after hatch were completely
protected against ciliostasis. It is concluded that IB M41-R has
great potential as vaccine to be administered in ovo.
All
publications mentioned in the above specification are herein
incorporated by reference. Various modifications and variations of
the described methods and system of the invention will be apparent
to those skilled in the art without departing from the scope and
spirit of the invention. Although the invention has been described
in connection with specific preferred embodiments, it should be
understood that the invention as claimed should not be unduly
limited to such specific embodiments. Indeed, various modifications
of the described modes for carrying out the invention which are
obvious to those skilled in molecular biology, virology or related
fields are intended to be within the scope of the following claims.
Claims
1.
A live, attenuated coronavirus comprising a variant replicase gene
encoding polyproteins comprising a mutation in one or both of
non-structural protein(s) nsp-10 and nsp-14, wherein the variant
replicase gene encodes a protein comprising an amino acid mutation
of Pro to Leu at the position corresponding to position 85 of SEQ ID
NO: 6, and/or wherein the variant replicase gene encodes a protein
comprising an amino acid mutation of Val to Leu at the position
corresponding to position 393 of SEQ ID NO: 7.
2.
The coronavirus according to claim 1 wherein the variant replicase
gene encodes a protein comprising one or more amino acid mutations
selected from:
- an amino acid mutation of Leu to Ile at the position corresponding to position 183 of SEQ ID NO: 8; and
- an amino acid mutation of Val to Ile at the position corresponding to position 209 of SEQ ID NO: 9.
3.
The coronavirus according to claim 1 wherein the replicase gene
encodes a protein comprising the amino acid mutations Val to Leu at
the position corresponding to position 393 of SEQ ID NO: 7; Leu to
Ile at the position corresponding to position 183 of SEQ ID NO: 8;
and Val to Ile at the position corresponding to position 209 of SEQ
ID NO: 9.
4.
The coronavirus according to claim 1 wherein the replicase gene
encodes a protein comprising the amino acid mutations Pro to Leu at
the position corresponding to position 85 of SEQ ID NO: 6; Val to
Leu at the position corresponding to position 393 of SEQ ID NO: 7;
Leu to Ile at the position corresponding to position 183 of SEQ ID
NO: 8; and Val to Ile at the position corresponding to position 209
of SEQ ID NO: 9.
5.
The coronavirus according to claim 1 wherein the replicase gene
comprises at least one nucleotide substitutions selected from:
compared to the sequence shown as SEQ ID NO: 1.
- C to Tat nucleotide position 12137; and
- G to C at nucleotide position 18114;
- compared to the sequence shown as SEQ ID NO: 1;
- and optionally, comprises one or more nucleotide substitutions selected from T to A at nucleotide position 19047; and
- G to A at nucleotide position 20139;
6.
The coronavirus according to claim 1 which is an infectious
bronchitis virus (IBV).
7.
The coronavirus according to claim 1 which is IBV M41.
8.
The coronavirus according to claim 7, which comprises an S protein
at least, part of which is from an IBV serotype other than M41.
9.
The coronavirus according to claim 8, wherein the S1 subunit is from
an IBV serotype other than M41.
10.
The coronavirus according to claim 8, wherein the S protein is from
an IBV serotype other than M41.
11.
The coronavirus according to claim 1 which has reduced pathogenicity
compared to a coronavirus expressing a corresponding wild-type
replicase, wherein the virus is capable of replicating without being
pathogenic to the embryo when administered to an embryonated egg.
12.
A variant replicase gene as defined in claim 1.
13.
A protein encoded by a variant coronavirus replicase gene according
to claim 12.
14.
A plasmid comprising a replicase gene according to claim 12.
15.
A method for making the coronavirus according to claim 1 which
comprises the following steps:
- (i) transfecting a plasmid according to claim 14 into a host cell;
- (ii) infecting the host cell with a recombining virus comprising the genome of a coronavirus strain with a replicase gene;
- (iii) allowing homologous recombination to occur between the replicase gene sequences in the plasmid and the corresponding sequences in the recombining virus genome to produce a modified replicase gene; and
- (iv) selecting for recombining virus comprising the modified replicase gene.
16.
The method according to claim 15, wherein the recombining virus is a
vaccinia virus.
17.
The method according to claim 15 which also includes the step:
- (v) recovering recombinant coronavirus comprising the modified replicase gene from the DNA from the recombining virus from step (iv).
18.
A cell capable of producing a coronavirus according to claim 1.
19.
A vaccine comprising a coronavirus according to claim 1 and a
pharmaceutically acceptable carrier.
20.
A method for treating and/or preventing a disease in a subject which
comprises the step of administering a vaccine according to claim 19
to the subject.
21.
The method of claim 20, wherein the disease is infectious bronchitis
(IB).
22.
The method according to claim 20 wherein the method of
administration is selected from the group consisting of; eye drop
administration, intranasal administration, drinking water
administration, post-hatch injection and in ovo injection.
23.
The method according to claim 21 wherein the administration is in
ovo vaccination.
24.
A method for producing a vaccine according to claim 19, which
comprises the step of infecting a cell according to claim 18 with a
coronavirus according to claim 1.
25.
The coronavirus according to claim 1, further comprising a mutation
in one or both of nsp-15 and nsp-16.
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Patent
History
Patent
number:
10130701Type: GrantFiled:
Jul 23, 2015Date
of Patent:
Nov 20, 2018Patent
Publication Number: 20170216427
Assignee: THE PIRBRIGHT INSTITUTE (Woking, Pirbright)Inventors: Erica Bickerton (Woking), Sarah Keep (Woking), Paul Britton (Woking)Primary Examiner: Bao Q Li
Application Number: 15/328,179
Assignee: THE PIRBRIGHT INSTITUTE (Woking, Pirbright)Inventors: Erica Bickerton (Woking), Sarah Keep (Woking), Paul Britton (Woking)Primary Examiner: Bao Q Li
Application Number: 15/328,179
Classifications
Current
U.S. Class: Coronaviridae
(e.g., Neonatal Calf Diarrhea Virus, Feline Infectious Peritonitis
Virus, Canine Coronavirus, Etc.) (424/221.1)
International Classification: A61K 39/215 (20060101); C12N 7/00 (20060101); C12N 9/12 (20060101); A61K 39/00 (20060101);
International Classification: A61K 39/215 (20060101); C12N 7/00 (20060101); C12N 9/12 (20060101); A61K 39/00 (20060101);
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