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US10130701B2 - CORONAVIRUS - GOOGLE PATENTS

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Publication number US10130701B2 US10130701B2 US15/328,179 US201515328179A
US10130701B2 US 10130701 B2 US10130701 B2 US 10130701B2 US 201515328179 A
US201515328179 A US 201515328179A US 10130701 B2 US10130701 B2 US 10130701B2
Authority US United States Prior art keywords coronavirus seq virus replicase
gene protein Prior art date 2014-07-23 Legal status (The legal status is an
assumption and is not a legal conclusion. Google has not performed a legal
analysis and makes no representation as to the accuracy of the status listed.)
Active Application number US15/328,179 Other versions US20170216427A1 (en
Inventor Erica Bickerton Sarah Keep Paul Britton Current Assignee (The listed
assignees may be inaccurate. Google has not performed a legal analysis and makes
no representation or warranty as to the accuracy of the list.) Pirbright
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analysis and makes no representation as to the accuracy of the date listed.)
2014-07-23 Filing date 2015-07-23 Publication date 2018-11-20 Family has
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ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors:
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CLASSIFICATIONS

* * A—HUMAN NECESSITIES
* A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
* A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
* A61K39/00—Medicinal preparations containing antigens or antibodies
* A61K39/12—Viral antigens
* A61K39/215—Coronaviridae, e.g. avian infectious bronchitis virus
* * C—CHEMISTRY; METALLURGY
* C07—ORGANIC CHEMISTRY
* C07K—PEPTIDES
* C07K14/00—Peptides having more than 20 amino acids; Gastrins;
Somatostatins; Melanotropins; Derivatives thereof
* C07K14/005—Peptides having more than 20 amino acids; Gastrins;
Somatostatins; Melanotropins; Derivatives thereof from viruses
* * A—HUMAN NECESSITIES
* A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
* A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL
PREPARATIONS
* A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
* A61P31/12—Antivirals
* A61P31/14—Antivirals for RNA viruses
* * C—CHEMISTRY; METALLURGY
* C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY;
MUTATION OR GENETIC ENGINEERING
* C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING,
PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING;
CULTURE MEDIA
* C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or
purification thereof
* * C—CHEMISTRY; METALLURGY
* C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY;
MUTATION OR GENETIC ENGINEERING
* C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING,
PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING;
CULTURE MEDIA
* C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for
preparing, activating, inhibiting, separating or purifying enzymes
* C12N9/10—Transferases (2.)
* C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g.
kinases (2.7)
* C12N9/1241—Nucleotidyltransferases (2.7.7)
* C12N9/127—RNA-directed RNA polymerase (2.7.7.48), i.e. RNA replicase
* * C—CHEMISTRY; METALLURGY
* C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY;
MUTATION OR GENETIC ENGINEERING
* C12Y—ENZYMES
* C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
* C12Y207/07—Nucleotidyltransferases (2.7.7)
* C12Y207/07048—RNA-directed RNA polymerase (2.7.7.48), i.e. RNA replicase
* * A—HUMAN NECESSITIES
* A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
* A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
* A61K39/00—Medicinal preparations containing antigens or antibodies
* A61K2039/51—Medicinal preparations containing antigens or antibodies
comprising whole cells, viruses or DNA/RNA
* A61K2039/525—Virus
* A61K2039/5254—Virus avirulent or attenuated
* * A—HUMAN NECESSITIES
* A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
* A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
* A61K39/00—Medicinal preparations containing antigens or antibodies
* A61K2039/54—Medicinal preparations containing antigens or antibodies
characterised by the route of administration
* * A—HUMAN NECESSITIES
* A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
* A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
* A61K39/00—Medicinal preparations containing antigens or antibodies
* A61K2039/70—Multivalent vaccine
* * C—CHEMISTRY; METALLURGY
* C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY;
MUTATION OR GENETIC ENGINEERING
* C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING,
PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING;
CULTURE MEDIA
* C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING,
PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING;
CULTURE MEDIA ssRNA viruses positive-sense
* C12N2770/00011—Details
* C12N2770/20011—Coronaviridae
* C12N2770/20021—Viruses as such, e.g. new isolates, mutants or their genomic
sequences
* * C—CHEMISTRY; METALLURGY
* C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY;
MUTATION OR GENETIC ENGINEERING
* C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING,
PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING;
CULTURE MEDIA
* C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING,
PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING;
CULTURE MEDIA ssRNA viruses positive-sense
* C12N2770/00011—Details
* C12N2770/20011—Coronaviridae
* C12N2770/20022—New viral proteins or individual genes, new structural or
functional aspects of known viral proteins or genes
* * C—CHEMISTRY; METALLURGY
* C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY;
MUTATION OR GENETIC ENGINEERING
* C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING,
PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING;
CULTURE MEDIA
* C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING,
PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING;
CULTURE MEDIA ssRNA viruses positive-sense
* C12N2770/00011—Details
* C12N2770/20011—Coronaviridae
* C12N2770/20034—Use of virus or viral component as vaccine, e.g.
live-attenuated or inactivated virus, VLP, viral protein
* * C—CHEMISTRY; METALLURGY
* C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY;
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* C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING,
PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING;
CULTURE MEDIA
* C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING,
PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING;
CULTURE MEDIA ssRNA viruses positive-sense
* C12N2770/00011—Details
* C12N2770/20011—Coronaviridae
* C12N2770/20051—Methods of production or purification of viral material
* * C—CHEMISTRY; METALLURGY
* C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY;
MUTATION OR GENETIC ENGINEERING
* C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING,
PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING;
CULTURE MEDIA
* C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING,
PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING;
CULTURE MEDIA ssRNA viruses positive-sense
* C12N2770/00011—Details
* C12N2770/20011—Coronaviridae
* C12N2770/20061—Methods of inactivation or attenuation
* C12N2770/20062—Methods of inactivation or attenuation by genetic
engineering
* * C—CHEMISTRY; METALLURGY
* C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY;
MUTATION OR GENETIC ENGINEERING
* C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING,
PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING;
CULTURE MEDIA
* C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING,
PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING;
CULTURE MEDIA ssRNA viruses positive-sense
* C12N2770/00011—Details
* C12N2770/20011—Coronaviridae
* C12N2770/20071—Demonstrated in vivo effect

DEFINITIONS

* 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.
* IBV Avian infectious bronchitis virus
* IB infectious bronchitis virus
* IBV 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.
* 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.
* 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.
* IBV vaccines which are not associated with these issues, in particular
vaccines which may be used for in ovo vaccination.
* 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.
* the present inventors 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.
* 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 replicase gene may comprise one or more nucleotide substitutions selected
from the list of:
* the coronavirus may be an infectious bronchitis virus (IBV).
* IBV infectious bronchitis virus
* the coronavirus may be IBV M41.
* the coronavirus may comprise an S protein at least part of which is from an
IBV serotype other than M41.
* the S1 subunit or the entire S protein may be 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.
* the present invention provides a variant replicase gene as defined in
connection with the first aspect of the invention.
* the present invention provides a protein encoded by a variant coronavirus
replicase gene according to the second aspect of the invention.
* the present invention provides a plasmid comprising a replicase gene
according to the second aspect of the invention.
* the present invention provides a method for making the coronavirus according
to the first aspect of the invention which comprises the following steps:
* the recombining virus may be a vaccinia virus.
* the method may also include the step:
* the present invention provides a cell capable of producing a coronavirus
according to the first aspect of the invention.
* the present invention provides a vaccine comprising a coronavirus according
to the first aspect of the invention and a pharmaceutically acceptable
carrier.
* 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 disease may be infectious bronchitis (IB).
* IB infectious bronchitis
* 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.
* Vaccination may be by in ova vaccination.
* 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.
* FIG. 1 Growth kinetics of M41-R-6 and M41-R-12 compared to M41-CK (M41 EP4)
on CK cells
* 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 Central 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. 10 Position of amino acid mutations in mutated nsp10, nsp14, nsp15 and
nsp16 sequences.
* 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).
* 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.
* IBV infectious bronchitis virus
* porcine coronavirus transmissible gastroenteritis, TGE
* 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.
* FIP feline infectious peritonitis
* CoV canine coronavirus
* MHV mouse hepatitis virus
* Coronaviruses are divided into four groups, as shown below:
* 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.
* 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.
* 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 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.
* severe respiratory distress may occur.
* respiratory distress, nephritis decrease in egg production, and loss of
internal egg quality and egg shell quality are common.
* 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.
* 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.
* S spike glycoprotein
* E small membrane protein
* M integral membrane protein
* N nucleocapsid protein
* 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.
* 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.
* 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-21TM). 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.
* the genome sequence of IBV M41-CK is provided as SEQ ID NO: 1.
* 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.
* Gene 1 in IBV encodes 15 (16 in other coronaviruses) non-structural proteins
(nsp2-16), which are associated with RNA replication and transcription.
* reporter protein is used herein to refer to the pp1a and pp1ab polyproteins
or individual nsp subunits.
* replicase gene is used herein to refer to a nucleic acid sequence which
encodes for replicase proteins.
* Nsp Protein Key features 1 conserveed within but not between coronavirus
genetic groups; potential regulatory functions in the host cell. 2
Dispensable for MHV and SARS-CoV replication in tissue culture 3 Acidic
domain; macro domain with ADRP and poly (ADP-ribose)-binding activities; one
or two ZBD- 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 form a hexadecameric complex 9 ssRNA-binding protein, dimer 10
RNA-binding protein, homododecamer, zinc-binding domain, known to interact
with nsp14 and nsp16 11 Unknown 12 RNA-dependent RNA polymerase 13
Zinc-binding domain, NTPase, dNT
* 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 ⁇ / ⁇ fold comprised of five ⁇ -helices, one 3 10 -helix and three
⁇ -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.
* 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.
* MT N7-methyltransferase
* 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 Mn 2+ ions and there was little activity in the presence of Mg 2+
and Ca 2+ . 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.
* genomic and protein sequences for nsp-10, -14, -15 and -16 are provided as
SEQ ID NO: 2-5 and 6-9, respectively.
* 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.
* Attenuated 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.
* 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.
* 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.
* 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.
* 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.
* 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.
* 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.
* the variant replicase of the present invention when expressed in a
coronavirus, may cause the virus to replicate at non-pathogenic levels in
ovo.
* 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.
* the embryo and hatched chicken 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.
* 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.
* 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).
* 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.
* 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.
* clinical symptoms for example wheezing, snicking, loss of ciliary activity
* 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.
* 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.
* 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.
* 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.
* 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 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.
* the variant replicase gene may also be defined at the nucleotide level.
* 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.
* 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:
* 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 comprise the substitution C12137T.
* the nucleotide sequence may comprise substitution G18114C.
* the nucleotide sequence may comprise the substitution T19047A.
* the nucleotide sequence may comprise the substitution G20139A.
* the nucleotide sequence may comprise the substitutions C12137T and G18114C.
* the nucleotide sequence may comprise the substitutions C12137T and T19047A.
* the nucleotide sequence may comprise the substitutions C12137T and G20139A.
* the nucleotide sequence may comprise the substitutions G18114C and T19047A.
* the nucleotide sequence may comprise the substitutions G18114C and G20139A.
* the nucleotide sequence may comprise the substitutions T19047A and G20139A.
* the nucleotide sequence may comprise the substitutions C12137T, G18114C and
T19047A.
* the nucleotide sequence may comprise the substitutions C12137T, T19047A and
G20139A.
* the nucleotide sequence may comprise the substitutions C12137T, G18114C and
G20139A.
* the nucleotide sequence may comprise the substitutions G18114C, T19047A and
G20139A.
* the nucleotide sequence may comprise the substitutions C12137T, G18114C,
T19047A and G20139A.
* 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.
* the nucleotide sequence may be codon optimised for production in the
host/host cell of choice.
* It may be isolated, or as part of a plasmid, virus or host cell.
* 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 may be used to express a protein in a host cell.
* 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.
* 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.
* rVV vaccinia virus
* the recombinant vaccinia virus may be made using a vaccinia-virus based
reverse genetics system.
* the present invention also provides a method for making a viral particle by:
* 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.
* 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.
* the above method optionally includes the step:
* 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.
* 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.
* 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)).
* modified replicase gene e.g. recombinant vaccinia virus, (rVV)
* rVV recombinant vaccinia virus
* the coronavirus may be used to infect a cell.
* Coronavirus particles may be harvested, for example from the supernatant, by
methods known in the art, and optionally purified.
* the cell may be used to produce the coronavirus particle.
* 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.
* 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.
* recombinant recombining virus e.g. vaccinia virus
* 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.
* 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.
* 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.
* 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.
* an effective amount of drug, compound, or pharmaceutical composition is an
amount sufficient to reduce the incidence of Infectious Bronchitis.
* 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.
* the coronavirus of the present invention may be used to treat and/or prevent
a disease.
* 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.
* 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.
* IB infectious bronchitis
* Porcine epidemic diarrhoea Transmissible gastroenteritis
* Mouse hepatitis virus Mouse hepatitis virus
* Porcine haemagglutinating encephalomyelitis Severe acute respiratory syndrome
(SARS); or Bluecomb disease.
* SARS Severe acute respiratory syndrome
* the disease may be infectious bronchitis.
* 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.
* 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.
* 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).
* NDV Newcastle disease virus
* 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.
* the subject may be a chick, chicken or mouse (such as a laboratory mouse,
e.g. transgenic mouse).
* 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.
* 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.
* 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 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.
* 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 ).
* 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
).
* 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).
* 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.
* 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.
* M41-K All four mutations, Nsp-10, -14, -15 & -16 repaired in M41-R
* 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
* results 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.
* 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.
* the genome associated with M41-R is a potential backbone genome for a
rationally attenuated IBV.
* 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 10 3.65 EID 50 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).
* EID 50 1 Route Day(s) Day(s) End Nr. of Treatment per of of of of of eggs per
Treatment Description dose Admin Admin Challenge 2 Study treatment T01 None
NA NA NA NA NA 30 T02 IB M41-R 10 4 In ovo 18 days At 21 days At 26 30 NTX
Saline NA In ovo embryo- of age, 20 days 30 nation chickens of age per group
1 Dose volume 0.1 ml, NA, not applicable. 2 10 3.65 EID 50 per dose.
* IB M41-R was safe in commercial eggs, generated protection against clinical
signs and to an extent against ciliostasis.
* IB M41-R has great potential as vaccine to be administered in ovo.

ABSTRACT

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.

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:
* * 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;
* Val to Ile at position 209 of SEQ ID NO: 9.

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 replicase gene may comprise one or more nucleotide substitutions selected
from the list of:
C to T at nucleotide position 12137;
G to C at nucleotide position 18114;
T to A at nucleotide position 19047; and
G to A at nucleotide position 20139;
compared to the sequence shown as SEQ ID NO: 1.
The coronavirus may be an infectious bronchitis virus (IBV).
The coronavirus may be IBV M41.
The coronavirus may comprise an S protein at least part of which is from an IBV
serotype other than M41.
For example, the S1 subunit or the entire S protein may be 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:
* * (i) transfecting a plasmid according to the fourth aspect of the invention
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.

The recombining virus may be a vaccinia virus.
The method may also include the step:
* * (v) recovering recombinant coronavirus comprising the modified replicase
gene from the DNA from the recombining virus from step (iv).

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.
Further aspects of the invention provide:
* * the vaccine according to the seventh aspect of the invention for use in
treating and/or preventing a disease in a subject.
* use of a coronavirus according to the first aspect of the invention in the
manufacture of a vaccine for treating and/or preventing a disease in a
subject.

The disease may be infectious bronchitis (IB).
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.
Vaccination may be by in ova vaccination.
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.
DESCRIPTION OF THE FIGURES
FIG. 1—Growth kinetics of M41-R-6 and M41-R-12 compared to M41-CK (M41 EP4) on
CK cells
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. 10—Position of amino acid mutations in mutated nsp10, nsp14, nsp15 and
nsp16 sequences.
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.
Coronavirus
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.
Coronaviruses are divided into four groups, as shown below:
* * Alpha
* Canine coronavirus (CCoV)
* Feline coronavirus (FeCoV)
* Human coronavirus 229E (HCoV-229E)
* Porcine epidemic diarrhoea virus (PEDV)
* Transmissible gastroenteritis virus (TGEV)
* Human Coronavirus NL63 (NL or New Haven)
* Beta
* Bovine coronavirus (BCoV)
* Canine respiratory coronavirus (CRCoV)—Common in SE Asia and Micronesia
* Human coronavirus OC43 (HCoV-OC43)
* Mouse hepatitis virus (MHV)
* Porcine haemagglutinating encephalomyelitis virus (HEV)
* Rat coronavirus (Roy). Rat Coronavirus is quite prevalent in Eastern
Australia where, as of March/April 2008, it has been found among native
and feral rodent colonies.
* (No common name as of yet) (HCoV-HKU1)
* Severe acute respiratory syndrome coronavirus (SARS-CoV)
* Middle East respiratory syndrome coronavirus (MERS-CoV)
* Gamma
* Infectious bronchitis virus (IBV)
* Turkey coronavirus (Bluecomb disease virus)
* Pheasant coronavirus
* Guinea fowl coronavirus
* Delta
* Bulbul coronavirus (BuCoV)
* Thrush coronavirus (ThCoV)
* Munia coronavirus (MuCoV)
* Porcine coronavirus (PorCov) HKU15

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.
IBV
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.
The genome sequence of IBV M41-CK is provided as SEQ ID NO: 1.

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

Replicase
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.
A summary of the functions of coronavirus nsp proteins is provided in Table 1.

TABLE 1 Nsp Protein Key features

1 Conserved within but not between coronavirus genetic groups; potential
regulatory functions in the host cell. 2 Dispensable for MHV and SARS-CoV
replication in tissue culture 3 Acidic domain; macro domain with ADRP and poly
(ADP-ribose)-binding activities; one or two ZBD- 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 form a hexadecameric
complex 9 ssRNA-binding protein, dimer 10 RNA-binding protein, homododecamer,
zinc-binding 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 and DNA helicase, RNA 5′-triphosphate 14 3′-to 5′ exoribonuclease,
zinc-binding domain and N7- 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.

(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

Reduced Pathogenicity
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.

TABLE 2 IBV severity limits based on clinical signs:

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.
Variant
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:

ALIPHATIC Non-polar G A P I L V Polar- uncharged C S T M N Q Polar- charged D E
K R AROMATIC 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:
* * 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;
* Val to Ile at position 209 of SEQ ID NO: 9.

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.
The variant replicase gene may also be defined at the nucleotide level.
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:
* * C to Tat nucleotide position 12137;
* G to C at nucleotide position 18114;
* T to A at nucleotide position 19047; and
* G to A at nucleotide position 20139;
compared to the sequence shown as SEQ ID NO: 1.

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:
* * C to Tat nucleotide position 12137;
* G to Cat nucleotide position 18114;
* T to A at nucleotide position 19047; and
* G to A at nucleotide position 20139;
compared to the sequence shown as SEQ ID NO: 1.

The nucleotide sequence may comprise the substitution C12137T.
The nucleotide sequence may comprise substitution G18114C.
The nucleotide sequence may comprise the substitution T19047A.
The nucleotide sequence may comprise the substitution G20139A.
The nucleotide sequence may comprise the substitutions C12137T and G18114C.
The nucleotide sequence may comprise the substitutions C12137T and T19047A.
The nucleotide sequence may comprise the substitutions C12137T and G20139A.
The nucleotide sequence may comprise the substitutions G18114C and T19047A.
The nucleotide sequence may comprise the substitutions G18114C and G20139A.
The nucleotide sequence may comprise the substitutions T19047A and G20139A.
The nucleotide sequence may comprise the substitutions C12137T, G18114C and
T19047A.
The nucleotide sequence may comprise the substitutions C12137T, T19047A and
G20139A.
The nucleotide sequence may comprise the substitutions C12137T, G18114C and
G20139A.
The nucleotide sequence may comprise the substitutions G18114C, T19047A and
G20139A.
The nucleotide sequence may comprise the substitutions C12137T, G18114C, T19047A
and G20139A.
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.
The nucleotide sequence may be codon optimised for production in the host/host
cell of choice.
It may be isolated, or as part of a plasmid, virus or host cell.
Plasmid
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.
Recombinant Vaccinia Virus
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.
In this respect, the present invention also provides a method for making a viral
particle by:
* * (i) transfecting a plasmid as described in the previous section 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;
* (iv) selecting for recombining virus comprising the modified replicase
gene.

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.
The above method optionally includes the step:
* * (v) recovery of recombinant coronavirus comprising the modified replicase
gene from the DNA from the recombining virus from step (iv).

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).
Cell
The coronavirus may be used to infect a cell.
Coronavirus particles may be harvested, for example from the supernatant, by
methods known in the art, and optionally purified.
The cell may be used to produce the coronavirus particle.
Thus the present invention also provides a method for producing a coronavirus
which comprises the following steps:
(i) infection of a cell with a coronavirus according to the invention;
(ii) allowing the virus to replicate in the cell; and
(iii) harvesting the progeny virus.
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.
Vaccine
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.
Vaccination Method
The coronavirus of the present invention may be used to treat and/or prevent a
disease.
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 disease may be infectious bronchitis.
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.
EXAMPLES Example 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).

TABLE 3 Non-Synonymous mutations identified in the Nsps of M41-R full-length
genome

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 following rIBVs were produced:
M41R-nsp10, 15rep—M41-R with the mutations in Nsp-10 and Nsp-15 repaired
M41R-nsp10, 14, 15rep—M41-R with mutations in Nsp-10, -14 and -15 repaired
M41R-nsp10, 14, 16rep—M41-R with mutations in Nsp-10, -14 and -16 repaired
M41R-nsp10, 15, 16rep—M41-R with mutations in Nsp-10, -15 and -16 repaired
M41-K—All four mutations, Nsp-10, -14, -15 & -16 repaired in M41-R
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. 11a-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).
In Ovo Vaccination in Commercial Broiler Eggs
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.

TABLE 4 Design of a hatchability, safety, efficacy study in commercial eggs

EID50 1 Route Day(s) Day(s) End Nr. of Treatment per of of of of eggs per
Treatment Description dose Admin Admin Challenge2 Study treatment T01 None NA NA
NA NA NA 30 T02 IB M41-R 104 In ovo 18 days At 21 days At 26 30 NTX Saline NA In
ovo embryo- of age, 20 days 30 nation chickens of age per group 1Dose volume 0.1
ml, NA, not applicable. 2103.65 EID50 per dose.

TABLE 5 Hatch percentages and clinical data before and after challenge in
commercial chickens, for design see Table 1.

Before After challenge challenge

Hatch/ Vital/ Deaths/ Symptoms/ Deaths/ Symptoms/ Treatment total total total
total total total

None 28/30 Euthanized directly after hatch for blood collection

IB M41-R 28/30 28/28 1/20 0/19 1/19 3/181, 7 Saline 29/30 29/29 1/20 0/19 0/19
19/191, 2, 3, 4, 5, 6, 7 1Disturbed respiratory system 2Whizzing 3Change of
voice 4Breathing difficult 5Swollen intra-orbital sinuses 6Uneven growth 7Weak

TABLE 6 Results of the ciliostasis test after challenge, for design see Table 1.

Treatment Protected/total Percentage protection Saline 0/19 0% IB M41R 5/18 28%

In Ovo Vaccination in Specific Pathogen-Free (SPF) Eggs
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.

TABLE 7 Design of a hatchability, safety, efficacy study in SPF eggs

EID50 1 Route Day Day End Nr. of Treatment per of of of of eggs per Treatment
Description dose Admin Admin Challenge2 Study treatment T01 IB M41-R 105 In ovo
18 days At 21 days At 26 40 embryo- of age days T04 Saline NA In ovo nation of
age 40 NTX NA NA NA NA 10 1Dose volume 0.1 ml, NA, not applicable. 2 Challenge
dose 103.3 EID50 in 0.2 ml.

TABLE 8 Hatch percentages and clinical data before and after challenge in SPF
chickens, for design see Table 7.

Before After challenge challenge

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 — — —

TABLE 9 Results of the ciliostasis test after challenge, for design see Table 7.

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 (25)

The invention claimed is:
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:
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;
compared to the sequence shown as SEQ ID NO: 1.
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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CITED BY (11)

* Cited by examiner, † Cited by third party Publication number Priority date
Publication date Assignee Title WO2021156878A2 (en) 2020-02-09 2021-08-12 Nlc
Pharma Ltd Rapid detection test for sars-cov-2 US11319325B1 (en) 2021-05-11
2022-05-03 Enanta Pharmaceuticals, Inc. Macrocyclic spiropyrrolidine derived
antiviral agents US11325916B1 (en) 2021-07-29 2022-05-10 Enanta Pharmaceuticals,
Inc. Spiropyrrolidine derived antiviral agents US11339170B1 (en) 2021-07-23
2022-05-24 Enanta Pharmaceuticals, Inc. Spiropyrrolidine derived antiviral
agents US11352363B1 (en) 2020-11-23 2022-06-07 Enanta Pharmaceuticals, Inc.
Spiropyrrolidine derived antiviral agents US11358953B2 (en) 2020-07-20
2022-06-14 Enanta Pharmaceuticals, Inc. Functionalized peptides as antiviral
agents US11384090B2 (en) 2020-11-23 2022-07-12 Enanta Pharmaceuticals, Inc.
Spiropyrrolidine derived antiviral agents WO2023049300A1 (en) * 2021-09-22
2023-03-30 Biomedit, Llc Methods of inhibiting diseases caused by respiratory
viruses US11858945B2 (en) 2021-11-12 2024-01-02 Enanta Pharmaceuticals, Inc.
Alkyne-containing antiviral agents US11912714B2 (en) 2021-11-12 2024-02-27
Enanta Pharmaceuticals, Inc. Spiropyrrolidine derived antiviral agents
US11919910B2 (en) 2021-11-12 2024-03-05 Enanta Pharmaceuticals, Inc.
Spiropyrrolidine derived antiviral agents

FAMILIES CITING THIS FAMILY (12)

* Cited by examiner, † Cited by third party Publication number Priority date
Publication date Assignee Title EP3589731A4 (en) * 2017-03-03 2020-12-30 Loyola
University of Chicago Coronaviruses, vaccines comprising the same, and methods
for preventing disease US11696947B2 (en) * 2018-10-31 2023-07-11 Boehringer
Ingelheim Vetmedica Gmbh H52 IBV vaccine with heterologous spike protein
KR102119875B1 (en) * 2019-03-19 2020-06-05 한국화학연구원 South Korean strain Middle
East Respiratory syndrome coronavirus Infectious mutated gene and use thereof
EP3966318A1 (en) * 2019-05-10 2022-03-16 Boehringer Ingelheim Vetmedica GmbH
Attenuated ibv with extended cell culture and tissue tropism CN111254155A (en) *
2020-01-25 2020-06-09 王跃驹 Method for expressing virus vaccine by using plant as
host WO2021195137A1 (en) * 2020-03-23 2021-09-30 Loyola University Of Chicago
Coronavirus vaccine compositions and methods of using same WO2021203236A1 (en) *
2020-04-07 2021-10-14 四川骋誉生物制品有限公司 Bat-derived coronavirus vaccine for
prevention of covid-19 CN112029781B (en) * 2020-08-14 2023-01-03 中山大学 Novel
coronavirus SARS-CoV-2 safety replicon system and application thereof
JP2024004496A (en) * 2020-10-14 2024-01-17 一般財団法人阪大微生物病研究会 knockout coronavirus
CN112592905B (en) * 2020-12-28 2023-07-28 广州达安基因股份有限公司 DNA polymerase mixture
for novel coronavirus detection CN114751964B (en) * 2020-12-29 2024-03-08
苏州方舟生物科技有限公司 Universal vaccine protein fragment of beta coronavirus, screening
method and application thereof CN113308440B (en) * 2021-05-26 2023-04-28 武汉大学
N7-methyltransferase defective coronavirus attenuated vaccine strain, and
preparation method and application thereof

CITATIONS (5)

* Cited by examiner, † Cited by third party Publication number Priority date
Publication date Assignee Title WO2004092360A2 (en) 2003-04-10 2004-10-28 Chiron
Corporation The severe acute respiratory syndrome coronavirus WO2005049814A2
(en) 2003-11-18 2005-06-02 Vironovative B.V. Novel atypical pneumonia-causing
virus WO2007078203A1 (en) 2006-01-03 2007-07-12 Norwex Holding As Anti-bacterial
micro-fibre and production thereof US7452542B2 (en) * 2004-05-21 2008-11-18
Vanderbilt University Live attenuated coronavirus vaccines WO2011004146A1 (en)
2009-07-07 2011-01-13 Institute For Animal Health Chimaeric protein

FAMILY CITES FAMILIES (4)

* Cited by examiner, † Cited by third party Publication number Priority date
Publication date Assignee Title US7445785B2 (en) 2003-03-03 2008-11-04 Intervet
International B.V. Infectious bronchitis virus with an altered spike gene
CN100413537C (en) * 2004-01-29 2008-08-27 国家人类基因组南方研究中心 SARS coronary virus full
virus vaccine CN102851257A (en) * 2012-08-27 2013-01-02 上海启盛生物科技有限公司 Attenuated
vaccine strain for avian infectious bronchitis virus and application thereof
CN103397002A (en) * 2013-08-07 2013-11-20 广东温氏食品集团股份有限公司 Attenuated method for
fowl infectious bronchitis viruses

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* 2014-07-23 GB GBGB1413020.7A patent/GB201413020D0/en not_active Ceased
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* 2015-07-23 AU AU2015293633A patent/AU2015293633B2/en not_active Ceased
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PATENT CITATIONS (5)

* Cited by examiner, † Cited by third party Publication number Priority date
Publication date Assignee Title WO2004092360A2 (en) 2003-04-10 2004-10-28 Chiron
Corporation The severe acute respiratory syndrome coronavirus WO2005049814A2
(en) 2003-11-18 2005-06-02 Vironovative B.V. Novel atypical pneumonia-causing
virus US7452542B2 (en) * 2004-05-21 2008-11-18 Vanderbilt University Live
attenuated coronavirus vaccines WO2007078203A1 (en) 2006-01-03 2007-07-12 Norwex
Holding As Anti-bacterial micro-fibre and production thereof WO2011004146A1 (en)
2009-07-07 2011-01-13 Institute For Animal Health Chimaeric protein

NON-PATENT CITATIONS (21)

* Cited by examiner, † Cited by third party Title Altschul et al., Basic local
alignment search tool. J. Mol. Biol. 215: 403-10 (1990). Ammayapppan et al.,
Identification of sequence changes responsible for the attenuation of avian
infectious bronchitis virus strain Arkansas DPI, Arch. Virol., 154(3):495-9
(2009). Anonymous: "EM_STD:KF377577", Oct. 30, 2013. Armesto et al., A
recombinant avian infectious bronchitis virus expressing a heterologous spike
gene belonging to the 4/91 serotype, PLoS One, 6(8):e24352 (2011). Armesto et
al., The replicase gene of avian coronavirus infectious bronchitis virus is a
determinant of pathogenicity, PLoS One, 4(10):e7384 (2009). Armesto et al.,
Transient dominant selection for the modification and generation of recombinant
infectious bronchitis coronaviruses, Methods Mol. Biol., 454:255-73 (2008).
Ausubel et al., Short Protocols in Molecular Biology, 4th edition, Chapter 18
(1999). Britton et al., Generation of a recombinant avian coronavirus infectious
bronchitis virus using transient dominant selection, J. Virol. Methods,
123(2):203-11 (2005). Britton et al., Modification of the avian coronavirus
infectious bronchitis virus for vaccine development, Bioeng. Bugs., 3(2):114-9
(2012). Casais et al., Recombinant avian infectious bronchitis virus expressing
a heterologous spike gene demonstrates that the spike protein is a determinant
of cell tropism, J. Virol., 77(16):9084-9 (2003). Casais et al., Reverse
genetics system for the avian coronavirus infectious bronchitis virus, J.
Virol., 75(24):12359-69 (2001). Cavanagh et al., Manipulation of the infectious
bronchitis coronavirus genome for vaccine development and analysis of the
accessory proteins, Vaccine, 25(30):5558-62 (2007). Devereux et al., A
comprehensive set of sequence analysis programms for the VAX. Nucl. Acids
Res.12: 387-95 (1984). International Preliminary Report on Patentability,
International Application No. PCT/GB2015/052124, dated Jan. 24, 2017.
International Search Report and Written Opinion, International Application No.
PCT/GB2015/052124, dated Oct. 9, 2015. Larkin et al., Clustal W and Clustal X
version 2.0, Bioinformatics, 23(21):2947-8 (2007). Menachery et al., Attenuation
and restoration of severe acute respiratory syndrome coronavirus mutant lacking
2′-o-methyltransferase activity, J. Virol., 88(8):4251-64 (2014). Sperry Journal
of Virology, 2005, vol. 79, No. 6, pp. 3391-3400. * Tatusova et al., BLAST 2
Sequences, a new tool for comparing protein and nucleotide sequences, FEMS
Microbiol. Lett., 174(2):247-50 (1999). Wang et al., Attenuation of porcine
reproductive and respiratory syndrome virus strain MN184 using chimeric
construction with vaccine sequence, Virology, 371(2):418-29 (2008). Wei et al.,
Development and characterization of a recombinant infectious bronchitis virus
expressing the ectodomain region of S1 gene of H120 strain, Appl. Microbiol.
Biotechnol., 98(4):1727-35 (2014).

CITED BY (11)

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Pharma Ltd Rapid detection test for sars-cov-2 US11358953B2 (en) 2020-07-20
2022-06-14 Enanta Pharmaceuticals, Inc. Functionalized peptides as antiviral
agents US11352363B1 (en) 2020-11-23 2022-06-07 Enanta Pharmaceuticals, Inc.
Spiropyrrolidine derived antiviral agents US11384090B2 (en) 2020-11-23
2022-07-12 Enanta Pharmaceuticals, Inc. Spiropyrrolidine derived antiviral
agents US11319325B1 (en) 2021-05-11 2022-05-03 Enanta Pharmaceuticals, Inc.
Macrocyclic spiropyrrolidine derived antiviral agents US11339170B1 (en)
2021-07-23 2022-05-24 Enanta Pharmaceuticals, Inc. Spiropyrrolidine derived
antiviral agents US11325916B1 (en) 2021-07-29 2022-05-10 Enanta Pharmaceuticals,
Inc. Spiropyrrolidine derived antiviral agents WO2023049300A1 (en) * 2021-09-22
2023-03-30 Biomedit, Llc Methods of inhibiting diseases caused by respiratory
viruses US11858945B2 (en) 2021-11-12 2024-01-02 Enanta Pharmaceuticals, Inc.
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US11919910B2 (en) 2021-11-12 2024-03-05 Enanta Pharmaceuticals, Inc.
Spiropyrrolidine derived antiviral agents

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