Attenuated Infectious Bronchitis Virus

The present invention provides a live, attenuated coronavirus comprising a mutation in non-structural protein nsp-3 and/or deletion of accessory proteins 3a and 3b. The coronavirus may be used as a vaccine for treating and/or preventing a disease, such as infectious bronchitis, in a subject.

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Description
FIELD OF THE INVENTION

The present invention relates to an attenuated coronavirus comprising a mutation 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 Coronavirinae 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 ovo vaccination as they cause reduced hatchability. A 70% reduction in hatchability is seen in some cases.

Attenuation following multiple passages 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 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 mutation in non-structural protein nsp-3 and/or deletion of accessory proteins 3a and/or 3b.

The mutation in nsp-3 may be in the adenosine diphosphate-ribose-1′-phosphatase (ADRP) region.

The modified nsp-3 gene may encode a protein comprising one or more amino acid mutations selected from the list of:

    • a) Asn (N) to Ala (A) at position 373 in SEQ ID NO: 6; and
    • b) Gly (G) to Ser (S) at position 379 in SEQ ID NO: 6.

The modified nsp-3 gene may encode a protein comprising the amino acid mutation Asn (N) to Ala (A) at position 373 in SEQ ID NO: 6.

The modified nsp-3 gene may encode a protein comprising the amino acid mutation Gly (G) to Ser (S) at position 379 in SEQ ID NO: 6.

The modified nsp-3 gene may comprise one or more nucleotide substitutions selected from the list of:

    • a) A to G at nucleotide position 1116 and A to C at nucleotide position 1117 compared to the sequence shown as SEQ ID NO: 5; and
    • b) G to A at nucleotide position 1138 compared to the sequence shown as SEQ ID NO: 5.

The live attenuated coronavirus may comprise a deletion from nucleotide position 37 to 384 of the sequence shown as SEQ ID NO: 2.

The coronavirus may be an infectious bronchitis virus (IBV).

The coronavirus may be IBV M41.

The coronavirus may comprise an S protein wherein 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 may have reduced pathogenicity compared to a wild-type coronavirus, 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 modified nsp-3 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 modified nsp-3 gene according to the second aspect of the invention.

In a fourth aspect, the present invention provides a plasmid comprising a modified nsp-3 gene according to the second aspect of the invention or a modified gene 3 with a deletion of accessory protein 3a and/or 3b.

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 nsp-3 gene and/or gene 3;
    • (iii) allowing homologous recombination to occur between the gene sequences in the plasmid and the corresponding sequences in the recombining virus genome to produce a modified gene; and
    • (iv) selecting for recombining virus comprising the modified gene.

The recombining virus may be a vaccinia virus.

The method may also include the step:

    • (v) recovering recombinant coronavirus comprising the modified 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 ovo 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—plasmid used to produce rIBV M41K-S-ADRP.

FIG. 2—plasmid used to produce rIBV M41K-A-ADRP.

FIG. 3—plasmid used to produce rIBV M41K-del3ab.

FIG. 4—A) Rales; B) Combined respiratory symptoms (wheezing and rales); C) Snicking and D) Ciliary activity in birds infected with rIBV M41K-S-ADRP, rIBV M41K-A-ADRP or rIBV M41K-del3ab compared to M41-K (all bar charts are shown as Mock, rIBV M41K-A-ADRP, rIBV M41K-S-ADRP, rIBV M41K-del3ab, M41-K from left to right).

FIG. 5—Summary of clinical signs, virus detection and isolation on day 4 post-infection.

FIG. 6—Summary of clinical signs, virus detection and isolation on day 6 post-infection.

FIG. 7—Growth kinetics of mutant M41K-S-ADRP 4, M41K-S-ADRP 2 (replicates of the S-ADRP mutation) and M41K-A-ADRP compared to wild type M41-K6 and M41-CK EP4 on CK cells

FIG. 8—Growth kinetics of mutant M41K-del3ab 10 and M41K-del3ab 1 compared to wild type M41-CK and M41-K on CK cells

DETAILED DESCRIPTION

The present invention provides a mutant coronavirus which has reduced pathogenicity compared to a corresponding wild type coronavirus.

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 (RCV). 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 recombinant 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 recombinant coronavirus comprises substantially the same nucleotide sequence as the wild-type coronavirus. For example, the recombinant coronavirus of the present invention may have at least 80%, 85%, 90%, 95%, 98% or 99% identity with the wild type coronavirus sequence.

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.

The recombinant 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 ACTTAAGATAGATATTAATATATATCTATCACACTAGCCTTGCGCTAGATTTCCAACTTAACAAAACGGACTTAAATACCTACAGCTGGTCCT CATAGGTGTTCCATTGCAGTGCACTTTAGTGCCCTGGATGGCACCTGGCCACCTGTCAGGTTTTTGTTATTAAAATCTTATTGTTGCTGGTAT CACTGCTTGTTTTGCCGTGTCTCACTTTATACATCCGTTGCTTGGGCTACCTAGTATCCAGCGTCCTACGGGCGCCGTGGCTGGTTCGAGTGC GAAGAACCTCTGGTTCATCTAGCGGTAGGCGGGTGTGTGGAAGTAGCACTTCAGACGTACCGGTTCTGTTGTGTGAAATACGGGGTCACCTCC CCCCACATACCTCTAAGGGCTTTTGAGCCTAGCGTTGGGCTACGTTCTCGCATAAGGTCGGCTATACGACGTTTGTAGGGGGTAGTGCCAAAC AACCCCTGAGGTGACAGGTTCTGGTGGTGTTTAGTGAGCAGACATACAATAGACAGTGACAACATGGCTTCAAGCCTAAAACAGGGAGTATCT CCCAAACTAAGGGATGTCATTCTTGTATCCAAAGACATTCCTGAACAACTTTGTGACGCTTTGTTTTTCTATACGTCACACAACCCTAAGGAT TACGCTGATGCTTTTGCAGTTAGGCAGAAGTTTGATCGTAATCTGCAGACTGGGAAACAGTTCAAATTTGAAACTGTGTGTGGTCTCTTCCTC TTGAAGGGAGTTGACAAAATAACACCTGGCGTCCCAGCAAAAGTCTTAAAAGCCACTTCTAAGTTGGCAGATTTAGAAGACATCTTTGGTGTC TCTCCCTTTGCAAGAAAATATCGTGAACTTTTGAAGACAGCATGCCAGTGGTCTCTTACTGTAGAAACACTGGATGCTCGTGCACAAACTCTT GATGAAATTTTTGACCCTACTGAAATACTTTGGCTTCAGGTGGCAGCAAAAATCCAAGTTTCGGCTATGGCGATGCGCAGGCTTGTTGGAGAA GTAACTGCAAAAGTCATGGATGCTTTGGGCTCAAATATGAGTGCTCTTTTCCAGATTTTTAAACAACAAATAGTCAGAATTTTTCAAAAAGCG CTGGCTATTTTTGAGAATGTGAGTGAATTACCACAGCGTATTGCAGCACTTAAGATGGCTTTTGCTAAGTGTGCCAAGTCCATTACTGTTGTG GTTATGGAGAGGACTCTAGTTGTTAGAGAGTTCGCAGGAACTTGTCTTGCAAGCATTAATGGTGCTGTTGCAAAATTCTTTGAAGAACTCCCA AATGGTTTCATGGGTGCTAAAATTTTCACTACACTTGCCTTCTTTAGGGAGGCTGCAGTGAAAATTGTGGATAACATACCAAATGCACCGAGA GGCACTAAAGGGTTTGAAGTCGTTGGTAATGCCAAAGGTACACAAGTTGTTGTGCGTGGCATGCGAAATGACTTAACACTGCTTGACCAAAAA GCTGAAATTCCTGTGGAGTCAGAAGGTTGGTCTGCAATTTTGGGTGGACATCTTTGCTATGTCTTTAAGAGTGGTGATCGCTTTTACGCGGCA CCTCTTTCAGGAAATTTTGCATTGCATGATGTGCATTGTTGTGAGCGTGTTGTCTGTCTTTCTGATGGTGTAACACCGGAGATAAATGATGGA CTTATTCTTGCAGCAATCTACTCTTCTTTTAGTGTCGCAGAACTTGTGGCAGCCATTAAAAGGGGTGAACCATTTAAGTTTCTGGGTCATAAA TTTGTGTATGCAAAGGATGCAGCAGTTTCTTTTACATTAGCGAAGGCTGCTACTATTGCAGATGTTTTGAAGCTGTTTCAATCAGCGCGTGTG AAAGTAGAAGATGTTTGGTCTTCACTTACTGAAAAGTCTTTTGAATTCTGGAGGCTTGCATATGGAAAAGTGCGTAATCTCGAAGAATTTGTT AAGACTTGTTTTTGTAAGGCTCAAATGGCGATTGTGATTTTAGCGACAGTGCTTGGAGAGGGCATTTGGCATCTTGTTTCGCAAGTCATCTAT AAAGTAGGTGGTCTTTTTACTAAAGTTGTTGACTTTTGTGAAAAATATTGGAAAGGTTTTTGTGCACAGTTGAAAAGAGCTAAGCTCATTGTC ACTGAAACCCTCTGTGTTTTGAAAGGAGTTGCACAGCATTGTTTTCAACTATTGCTGGATGCAATACAGTTTATGTATAAAAGTTTTAAGAAG TGTGCACTTGGTAGAATCCATGGAGACTTGCTCTTCTGGAAAGGAGGTGTGCACAAAATTATTCAAGAGGGCGATGAAATTTGGTTTGACGCC ATTGATAGTATTGATGTTGAAGATCTGGGTGTTGTTCAAGAAAAATTGATTGATTTTGATGTTTGTGATAATGTGACACTTCCAGAGAACCAA CCCGGTCATATGGTTCAAATCGAGGATGACGGAAAGAACTACATGTTCTTCCGCTTCAAAAAGGATGAGAACATTTATTATACACCAATGTCA CAGCTTGGTGCTATTAATGTGGTTTGCAAAGCAGGCGGTAAAACTGTCACCTTTGGAGAAACTACTGTGCAAGAAATACCACCACCTGATGTT GTGTTTATTAAGGTTAGCATTGAGTGTTGTGGTGAACCATGGAATACAATCTTCAAAAAGGCTTATAAGGAGCCCATTGAAGTAGAGACAGAC CTCACAGTTGAACAATTGCTCTCTGTGGTCTATGAGAAAATGTGTGATGATCTCAAGCTGTTTCCGGAGGCTCCAGAACCACCACCATTTGAG AATGTCACACTTGTTGATAAGAATGGTAAAGATTTGGATTGCATAAAATCATGCCATCTGATCTATCGTGATTATGAGAGCGATGATGACATC GAGGAAGAAGATGCAGAAGAATGTGACACGGATTCAGGTGATGCTGAGGAGTGTGACACTAATTCAGAATGTGAAGAAGAAGATGAGGATACT AAAGTGTTGGCTCTTATACAAGACCCGGCAAGTAACAAATATCCTCTGCCTCTTGATGATGATTATAGCGTCTACAATGGATGTATTGTTCAT AAGGACGCTCTCGATGTTGTGAATTTACCATCTGGTGAAGAAACCTTTGTTGTCAATAACTGCTTTGAAGGGGCTGTTAAAGCTCTTCCGCAG AAAGTTATTGATGTTCTAGGTGACTGGGGTGAGGCTGTTGATGCGCAAGAACAATTGTGTCAACAAGAATCAACTCGGGTCATATCTGAGAAA TCAGTTGAGGGTTTTACTGGTAGTTGTGATGCAATGGCTGAACAAGCTATTGTTGAAGAGCAGGAAATAGTACCTGTTGTTGAACAAAGTCAG GATGTAGTTGTTTTTACACCTGCAGACCTAGAAGTTGTTAAAGAAACAGCAGAAGAGGTTGATGAGTTTATTCTCATTTCTGCTGTCCCTAAA GAAGAAGTTGTGTCTCAGGAGAAAGAGGAGCCACAGGTTGAGCAAGAGCCTACCCTAGTTGTTAAAGCACAACGTGAGAAGAAGGCTAAAAAG TTCAAAGTTAAACCAGCTACATGTGAAAAACCCAAATTTTTGGAGTACAAAACATGTGTGGGTGATTTGGCTGTTGTAATTGCCAAAGCATTG GATGAGTTTAAAGAGTTCTGCATTGTAAACGCTGCAAATGAGCACATGTCGCATGGTGGTGGCGTTGCAAAGGCAATTGCAGACTTTTGTGGA CCGGACTTTGTTGAATATTGCGCGGACTATGTTAAGAAACATGGTCCACAGCAAAAACTTGTCACACCTTCATTTGTTAAAGGCATTCAATGT GTGAATAATGTTGTAGGACCTCGCCATGGAGACAGCAACTTGCGTGAGAAGCTTGTTGCTGCTTACAAGAGTGTTCTTGTAGGTGGAGTGGTT AACTATGTTGTGCCAGTTCTCTCATCAGGGATTTTTGGTGTAGATTTTAAAATATCAATAGATGCTATGCGCGAAGCTTTTAAAGGTTGTGCC ATACGCGTTCTTTTATTTTCTCTGAGTCAAGAACACATCGATTATTTCGATGCAACTTGTAAGCAGAAGACAATTTATCTTACGGAGGATGGT GTTAAATACCGCTCTGTTGTTTTAAAACCTGGTGATTCTTTGGGTCAATTTGGACAGGTTTTTGCAAGAAATAAGGTAGTCTTTTCGGCTGAT GATGTTGAGGATAAAGAAATCCTCTTTATACCCACAACTGACAAGACTATTCTTGAATATTATGGTTTAGATGCGCAAAAGTATGTAACATAT TTGCAAACGCTTGCGCAGAAATGGGATGTTCAATATAGAGACAATTTTGTTATATTAGAGTGGCGTGACGGAAATTGCTGGATTAGTTCAGCA ATAGTTCTCCTTCAAGCTGCTAAAATTAGATTTAAAGGTTTTCTTGCAGAAGCATGGGCTAAACTGTTGGGTGGAGATCCTACAGACTTTGTT GCCTGGTGTTATGCAAGTTGCAATGCTAAAGTAGGTGATTTTTCAGATGCTAATTGGCTTTTGGCCAATTTAGCAGAACATTTTGACGCAGAT TACACAAATGCACTTCTTAAGAAGTGTGTGTCGTGCAATTGTGGTGTTAAGAGTTATGAACTTAGGGGTCTTGAAGCCTGTATTCAGCCAGTT CGAGCACCTAATCTTCTACATTTTAAAACGCAATATTCAAATTGCCCAACCTGTGGTGCAAGTAGTACGGATGAAGTAATAGAAGCTTCATTA CCGTACTTATTGCTTTTTGCTACTGATGGTCCTGCTACAGTTGATTGTGATGAAAATGCTGTAGGGACTGTTGTTTTCATTGGCTCTACTAAT AGTGGCCATTGTTATACACAAGCCGATGGTAAGGCTTTTGACAATCTTGCTAAGGATAGAAAATTTGGAAGGAAGTCGCCTTACATTACAGCA ATGTATACACGTTTTTCTCTTAGGAGTGAAAATCCCCTACTTGTTGTTGAACATAGTAAGGGTAAAGCTAAAGTAGTAAAAGAAGATGTTTCT AACCTTGCTACTAGTTCTAAAGCCAGTTTTGACGATCTTACTGACTTTGAACAGTGGTATGATAGCAACATCTATGAGAGTCTTAAAGTGCAG GAGACACCTGATAATCTTGATGAATATGTGTCATTTACGACAAAGGAAGATTCTAAGTTGCCACTGACACTTAAAGTTAGAGGTATCAAATCA GTTGTTGACTTTAGGTCTAAGGATGGTTTTACTTATAAGTTAACACCTGATACTGATGAAAATTCAAAAACACCAGTCTACTACCCAGTCTTG GATTCTATTAGTCTTAGGGCAATATGGGTTGAAGGCAGTGCTAATTTTGTTGTTGGGCATCCAAATTATTATAGTAAGTCTCTCCGAATTCCC ACGTTTTGGGAAAATGCCGAGAGCTTTGTTAAAATGGGTTATAAAATTGATGGTGTAACTATGGGCCTTTGGCGTGCAGAACACCTTAATAAA CCTAATTTGGAGAGAATTTTTAACATTGCTAAGAAAGCTATTGTTGGATCTAGTGTTGTTACTACGCAGTGTGGTAAAATACTAGTTAAAGCA GCTACATACGTTGCCGATAAAGTAGGTGATGGTGTAGTTCGCAATATTACAGATAGAATTAAGGGTCTTTGTGGATTCACACGTGGCCATTTT GAAAAGAAAATGTCCCTACAATTTCTAAAGACACTTGTGTTCTTTTTCTTTTATTTCTTAAAGGCTAGTGCTAAGAGTTTAGTTTCTAGCTAT AAGATTGTGTTATGTAAGGTGGTGTTTGCTACCTTACTTATAGTGTGGTTTATATACACAAGTAATCCAGTAGTGTTTACTGGAATACGTGTG CTAGACTTCCTATTTGAAGGTTCTTTATGTGGTCCTTATAATGACTACGGTAAAGATTCTTTTGATGTGTTACGCTATTGTGCAGGTGATTTT ACTTGTCGTGTGTGTTTACATGATAGAGATTCACTTCATCTGTACAAACATGCTTATAGCGTAGAACAAATTTATAAGGATGCAGCTTCTGGC ATTAACTTTAATTGGAATTGGCTTTATTTGGTCTTTCTAATATTATTTGTTAAGCCAGTGGCAGGTTTTGTTATTATTTGTTATTGTGTTAAG TATTTGGTATTGAGTTCAACTGTGTTGCAAACTGGTGTAGGTTTTCTAGATTGGTTTGTAAAAACAGTTTTTACCCATTTTAATTTTATGGGA GCGGGATTTTATTTCTGGCTCTTTTACAAGATATACGTACAAGTGCATCATATATTGTACTGTAAGGATGTAACATGTGAAGTGTGCAAGAGA GTTGCACGCAGCAACAGGCAAGAGGTTAGCGTTGTAGTTGGTGGACGCAAGCAAATAGTGCATGTTTACACTAATTCTGGCTATAACTTTTGT AAGAGACATAATTGGTATTGTAGAAATTGTGATGATTATGGTCACCAAAATACATTTATGTCCCCTGAAGTTGCTGGCGAGCTTTCTGAAAAG CTTAAGCGCCATGTTAAACCTACAGCATATGCTTACCACGTTGTGTATGAGGCATGCGTGGTTGATGATTTTGTTAATTTAAAATATAAGGCT GCAATTCCTGGTAAGGATAATGCATCTTCTGCTGTTAAGTGTTTCAGTGTTACAGATTTTTTAAAGAAAGCTGTTTTTCTTAAGGAGGCATTG AAATGTGAACAAATATCTAATGATGGTTTTATAGTGTGTAATACACAGAGTGCGCATGCACTAGAGGAAGCAAAGAATGCAGCCGTCTATTAT GCGCAATATCTGTGTAAGCCAATACTTATACTTGACCAGGCACTTTATGAGCAATTAATAGTAGAGCCTGTGTCTAAGAGTGTTATAGATAAA GTGTGTAGCATTTTGTCTAATATAATATCTGTAGATACTGCAGCTTTAAATTATAAGGCAGGCACACTTCGTGATGCTCTGCTTTCTATTACT AAAGACGAAGAAGCCGTAGATATGGCTATCTTCTGCCACAATCATGAAGTGGAATACACTGGTGACGGTTTTACTAATGTGATACCGTCATAT GGTATGGACACTGATAAGTTGACACCTCGTGATAGAGGGTTTTTGATAAATGCAGATGCTTCTATTGCTAATTTAAGAGTCAAAAATGCTCCT CCGGTAGTATGGAAGTTTTCTGATCTTATTAAATTGTCTGACAGTTGCCTTAAATATTTAATTTCAGCTACTGTCAAGTCAGGAGGTCGTTTC TTTATAACAAAGTCTGGTGCTAAACAAGTTATTTCTTGTCATACCCAGAAACTGTTGGTAGAGAAAAAGGCAGGTGGTGTTATTAATAACACT TTTAAATGGTTTATGAGTTGTTTTAAATGGCTTTTTGTCTTTTATATACTTTTTACAGCATGTTGTTTGGGTTACTACTATATGGAGATGAAT AAAAGTTTTGTTCACCCCATGTATGATGTAAACTCCACACTGCATGTTGAAGGGTTCAAAGTTATAGACAAAGGTGTTATTAGAGAGATTGTG TCAGAAGATAATTGTTTCTCTAATAAGTTTGTTAATTTTGACGCCTTTTGGGGTAAATCATATGAAAATAATAAAAACTGTCCAATTGTTACA GTTGTTATAGATGGTGACGGGACAGTAGCTGTTGGTGTTCCTGGTTTTGTATCATGGGTTATGGATGGTGTTATGTTTGTGCATATGACACAG ACTGATCGTAGACCTTGGTACATTCCTACCTGGTTTAATAGAGAAATTGTTGGTTACACTCAGGATTCAATTATCACTGAGGGTAGTTTTTAT ACATCTATAGCATTATTTTCTGCTAGATGTTTATATTTAACAGCCAGCAATACACCTCAATTGTATTGTTTTAATGGCGACAATGATGCACCT GGAGCCTTACCATTTGGTAGTATTATTCCTCATAGAGTATACTTCCAACCTAATGGTGTTAGGCTTATAGTTCCACAACAAATACTGCATACA CCCTACATAGTGAAGTTTGTTTCAGACAGCTATTGTAGAGGTAGTGTATGTGAGTATACTAAACCAGGTTACTGTGTGTCACTAGACTCCCAA TGGGTTTTGTTTAATGATGAATACATTAGTAAACCTGGCGTTTTCTGTGGTTCTACTGTTAGAGAACTTATGTTTAATATGGTTAGTACATTC TTTACTGGTGTCAACCCTAATATTTATATTCAGCTAGCAACTATGTTTTTAATACTAGTTGTTATTGTGTTAATTTTTGCAATGGTTATAAAG TTTCAAGGTGTTTTTAAAGCTTATGCGACCATTGTGTTTACAATAATGTTAGTTTGGGTTATTAATGCATTTGTTTTGTGTGTACATAGTTAT AATAGTGTTTTAGCTGTTATATTATTAGTACTCTATTGCTATGCATCATTGGTTACAAGTCGCAATACTGCTATAATAATGCATTGTTGGCTT GTTTTTACCTTTGGTTTAATAGTACCCACATGGTTGGCTTGTTGCTATCTGGGATTTATTCTTTATATGTACACACCGTTGGTTTTCTGGTGT TACGGTACTACTAAAAATACTCGTAAGTTGTATGATGGCAACGAGTTTGTTGGTAATTATGACCTTGCTGCGAAGAGCACTTTTGTTATTCGT GGTACTGAATTTGTTAAGCTTACGAATGAGATAGGTGATAAATTTGAAGCCTATCTTTCTGCGTATGCTAGACTTAAATACTATTCAGGCACT GGTAGTGAGCAAGATTACTTGCAAGCTTGTCGTGCATGGTTAGCTTATGCTTTGGACCAATATAGAAATAGTGGTGTTGAGGTTGTTTATACC CCACCGCGTTACTCTATTGGTGTTAGTAGACTACACGCTGGTTTTAAAAAACTAGTTTCTCCTAGTAGTGCTGTTGAGAAGTGCATTGTTAGT GTCTCTTATAGAGGCAATAATCTTAATGGACTGTGGCTGGGTGATTCTATTTACTGCCCACGCCATGTGTTAGGTAAGTTTAGTGGTGACCAG TGGGGTGACGTACTAAACCTTGCTAATAATCATGAGTTTGAAGTTGTAACTCAAAATGGTGTTACTTTGAATGTTGTCAGCAGGCGGCTTAAA GGAGCAGTTTTAATTTTACAAACTGCAGTTGCCAATGCTGAAACTCCTAAGTATAAGTTTGTTAAAGCTAATTGTGGTGATAGTTTCACTATA GCTTGTTCTTATGGTGGTACAGTTATAGGACTTTACCCTGTCACTATGCGTTCTAATGGTACTATTAGAGCATCTTTCCTAGCAGGAGCCTGT GGCTCAGTTGGTTTTAATATAGAAAAGGGTGTAGTTAATTTCTTTTATATGCACCATCTTGAGTTACCTAATGCATTACACACTGGAACTGAC CTAATGGGTGAGTTTTATGGTGGTTATGTAGATGAAGAGGTTGCGCAAAGAGTGCCACCAGATAATCTAGTTACTAACAATATTGTAGCATGG CTCTATGCGGCAATTATTAGTGTTAAAGAAAGTAGTTTTTCACAACCTAAATGGTTGGAGAGTACTACTGTTTCTATTGAAGATTACAATAGG TGGGCTAGTGATAATGGTTTTACTCCATTTTCCACTAGTACTGCTATTACTAAATTAAGTGCTATAACTGGGGTTGATGTTTGTAAACTCCTT CGCACTATTATGGTAAAAAGTGCTCAATGGGGTAGTGATCCCATTTTAGGACAATATAATTTTGAAGACGAATTGACACCAGAATCTGTATTT AATCAAGTTGGTGGTGTTAGGTTACAGTCTTCTTTTGTAAGAAAAGCTACATCTTGGTTTTGGAGTAGATGTGTATTAGCTTGCTTCTTGTTT GTGTTGTGTGCTATTGTCTTATTTACGGCAGTGCCACTTAAGTTTTATGTACATGCAGCTGTTATTTTGTTGATGGCTGTGCTCTTTATTTCT TTTACTGTTAAACATGTTATGGCATACATGGACACTTTCCTATTGCCTACATTGATTACAGTTATTATTGGAGTTTGTGCTGAAGTCCCTTTC ATATACAATACTCTAATTAGTCAAGTTGTTATTTTCTTAAGCCAATGGTATGATCCTGTAGTCTTTGATACTATGGTACCATGGATGTTATTG CCATTAGTGTTGTACACTGCTTTTAAGTGTGTACAAGGCTGCTATATGAATTCTTTCAATACTTCTTTGTTAATGCTGTATCAGTTTATGAAG TTAGGTTTTGTTATTTACACCTCTTCAAACACTCTTACTGCATATACAGAAGGTAATTGGGAGTTATTCTTTGAGTTGGTTCACACTATTGTG TTGGCTAATGTTAGTAGTAATTCCTTAATTGGTTTAATTGTTTTTAAGTGTGCTAAGTGGATTTTATATTATTGCAATGCAACATACTTTAAT AATTATGTGTTAATGGCAGTCATGGTTAATGGCATAGGCTGGCTTTGCACCTGTTACTTTGGATTGTATTGGTGGGTTAATAAAGTTTTTGGT TTAACCTTAGGTAAATACAATTTTAAAGTTTCAGTAGATCAATATAGGTATATGTGTTTGCATAAGGTAAATCCACCTAAAACTGTGTGGGAG GTCTTTACTACAAATATACTTATACAAGGAATTGGAGGCGATCGTGTGTTGCCTATAGCTACAGTGCAATCTAAATTGAGTGATGTAAAGTGT ACAACTGTTGTTTTAATGCAGCTTTTGACTAAGCTTAATGTTGAAGCAAATTCAAAAATGCATGCTTATCTTGTTGAGTTACACAATAAAATC CTCGCATCTGATGATGTTGGAGAGTGCATGGATAATTTATTGGGTATGCTTATAACACTATTTTGTATAGATTCTACTATTGATTTGGGTGAG TATTGTGATGATATACTTAAGAGGTCAACTGTATTACAATCGGTTACTCAAGAGTTTTCGCACATACCCTCGTATGCTGAATATGAAAGAGCT AAGAGTATTTATGAAAAGGTTTTAGCCGATTCTAAAAATGGTGGTGTAACACAGCAAGAGCTTGCTGCATATCGTAAAGCTGCCAATATTGCA AAGTCAGTTTTTGATAGAGACTTGGCTGTTCAAAAGAAGTTAGATAGCATGGCAGAACGTGCTATGACAACAATGTATAAAGAGGCGCGTGTA ACTGATAGAAGAGCAAAATTAGTTTCATCATTACATGCACTACTTTTTTCAATGCTTAAGAAAATAGATTCTGAGAAGCTTAATGTCTTATTT GACCAGGCGAATAGTGGTGTTGTACCCCTAGCAACTGTTCCAATTGTTTGTAGTAATAAGCTTACCCTTGTTATACCAGACCCAGAGACGTGG GTCAAGTGTGTGGAGGGTGTGCATGTTACATATTCAACAGTTGTTTGGAATATAGACTGTGTTACTGATGCCGATGGCACAGAGTTACACCCC ACTTCTACAGGTAGTGGATTGACTTACTGTATAAGTGGTGATAATATAGCATGGCCTTTAAAGGTTAACTTGACTAGGAATGGGCATAATAAG GTTGATGTTGCCTTGCAAAATAATGAGCTTATGCCTCACGGTGTAAAGACAAAGGCTTGCGTAGCAGGTGTAGATCAAGCACATTGTAGCGTT GAGTCTAAATGTTATTATACAAGTATTAGTGGCAGTTCAGTTGTAGCTGCTATTACCTCTTCAAATCCTAATCTGAAAGTAGCCTCTTTTTTG AATGAGGCAGGTAATCAGATTTATGTAGACTTAGACCCACCATGTAAATTTGGTATGAAAGTGGGTGATAAGGTTGAAGTTGTTTACCTGTAT TTTATAAAAAATACGAGGTCTATTGTAAGAGGTATGGTACTTGGTGCTATATCTAATGTTGTTGTGTTACAATCTAAAGGTCATGAGACAGAG GAAGTGGATGCTGTAGGCATTCTCTCACTTTGTTCTTTTGCAGTAGATCCTGCGGATACATATTGTAAATATGTGGCAGCAGGTAATCAACCT TTAGGTAACTGTGTTAAAATGTTGACAGTACATAATGGTAGTGGTTTTGCAATAACATCAAAGCCAAGTCCAACTCCGGATCAGGATTCTTAT GGAGGAGCTTCTGTGTGTCTTTATTGTAGAGCACATATAGCACACCCTGGCGGAGCAGGAAATTTAGATGGACGCTGTCAATTTAAAGGTTCT TTTGTGCAAATACCTACTACGGAGAAAGATCCTGTTGGATTCTGTCTACGTAACAAGGTTTGCACTGTTTGTCAGTGTTGGATTGGTTATGGA TGTCAGTGTGATTCACTTAGACAACCTAAACCTTCTGTTCAGTCAGTTGCTGTTGCATCTGGTTTTGATAAGAATTATTTAAACGGGTACGGG GTAGCAGTGAGGCTCGGCTGATACCCCTAGCTAATGGATGTGACCCCGATGTTGTAAAGCGAGCCTTTGATGTTTGTAATAAGGAATCAGCCG GTATGTTTCAAAATTTGAAGCGTAACTGTGCACGATTCCAAGAAGTACGTGATACTGAAGATGGAAATCTTGAGTATTGTGATTCTTATTTTG TGGTTAAACAAACCACTCCTAGTAATTATGAACATGAGAAAGCTTGTTATGAAGACTTAAAGTCAGAAGTAACAGCTGATCATGATTTCTTTG TGTTCAATAAGAACATTTATAATATTAGTAGGCAGAGGCTTACTAAGTATACTATGATGGATTTTTGCTATGCTTTGCGGCACTTTGACCCAA AGGATTGCGAAGTTCTTAAAGAAATACTTGTCACTTATGGTTGTATAGAAGATTATCACCCTAAGTGGTTTGAAGAGAATAAGGATTGGTACG ACCCAATAGAAAACCCTAAATATTATGCCATGTTGGCTAAAATGGGACCTATTGTACGACGTGCTTTATTGAATGCTATTGAGTTCGGAAACC TCATGGTTGAAAAAGGTTATGTTGGTGTTATTACACTTGATAACCAAGATCTTAATGGCAAATTTTATGATTTTGGTGATTTTCAGAAGACAG CGCCTGGTGCTGGTGTTCCTGTTTTTGATACGTATTATTCTTACATGATGCCCATCATAGCCATGACTGATGCGTTGGCACCTGAGAGGTATT TTGAATATGATGTGCATAAGGGTTATAAATCTTATGATCTCCTCAAGTATGATTATACTGAGGAGAAACAAGATTTGTTTCAGAAGTACTTTA AGTATTGGGATCAAGAGTATCACCCTAACTGTCGCGACTGTAGTGATGACAGGTGTTTGATACATTGTGCAAACTTCAACATCTTGTTTTCTA CACTTGTACCGCAGACTTCTTTCGGTAATTTGTGTAGAAAGGTTTTTGTTGATGGTGTACCATTTATAGCTACTTGTGGCTATCATTCTAAGG AACTTGGTGTTATTATGAATCAAGATAACACCATGTCATTTTCAAAAATGGGTTTGAGTCAACTCATGCAGTTTGTTGGAGATCCTGCCTTGT TAGTGGGGACATCCAATAAATTAGTGGATCTTAGAACGTCTTGTTTTAGTGTTTGTGCTTTAGCGTCTGGTATTACTCATCAAACGGTAAAAC CAGGTCACTTTAACAAGGATTTCTACGATTTTGCAGAGAAGGCTGGTATGTTTAAGGAAGGTTCTTCTATACCACTTAAACATTTCTTCTACC CACAGACTGGTAATGCTGCTATAAACGATTATGATTATTATCGTTATAACAGGCCTACCATGTTTGATATACGTCAACTTTTATTTTGTTTAG AAGTGACTTCTAAATATTTTGAATGTTATGAAGGCGGCTGTATACCAGCAAGCCAAGTTGTAGTTAACAATTTAGATAAGAGTGCAGGTTATC CGTTCAATAAGTTTGGAAAGGCCCGTCTCTATTATGAAATGAGTCTAGAGGAGCAGGACCAACTCTTTGAGAGTACAAAGAAGAACGTCCTGC CTACTATAACTCAGATGAATTTAAAATATGCCATATCCGCGAAAAATAGAGCGCGTACAGTGGCAGGTGTGTCTATCCTTTCTACTATGACTA ATAGGCAGTTTCATCAGAAGATTCTTAAGTCTATAGTCAACACTAGAAACGCTCCTGTAGTTATTGGAACAACCAAGTTTTATGGCGGTTGGG ATAACATGTTGAGAAACCTTATTCAGGGTGTTGAAGACCCGATTCTTATGGGTTGGGATTATCCAAAGTGTGATAGAGCAATGCCTAATTTGT TGCGTATAGCAGCATCTTTAGTACTCGCTCGTAAACACACTAATTGTTGTACTTGGTCTGAACGCGTTTATAGGTTGTATAATGAATGCGCTC AGGTTTTATCTGAAACTGTCTTAGCTACAGGTGGTATATATGTGAAACCTGGTGGTACTAGCAGTGGAGATGCTACTACTGCTTATGCAAACA GTGTTTTCAACATAATACAAGCCACATCTGCTAATGTTGCGCGTCTTTTGAGTGTTATAACGCGTGATATTGTATATGATGACATTAAGAGCT TGCAGTATGAATTGTACCAGCAGGTTTATAGGCGAGTCAATTTTGACCCAGCATTTGTTGAAAAGTTTTATTCTTATTTGTGTAAGAATTTCT CATTGATGATCTTGTCTGACGACGGTGTTGTTTGTTATAACAACACATTAGCCAAACAAGGTCTTGTAGCAGATATTTCTGGTTTTAGAGAAG TTCTCTACTATCAGAACAATGTTTTTATGGCTGATTCTAAATGTTGGGTTGAACCAGATTTAGAAAAAGGCCCACATGAATTTTGTTCACAGC ACACAATGTTAGTGGAGGTTGATGGTGAGCCTAGATACTTGCCATATCCAGACCCATCACGTATTTTGTGTGCATGTGTTTTTGTAGATGATT TGGATAAGACAGAATCTGTGGCTGTTATGGAGCGTTATATCGCTCTTGCCATAGATGCGTACCCACTAGTACATCATGAAAATGAGGAGTACA AGAAGGTATTCTTTGTGCTTCTTTCATACATCAGAAAACTCTATCAAGAGCTTTCTCAGAATATGCTTATGGACTACTCTTTTGTAATGGATA TAGATAAGGGTAGTAAATTTTGGGAACAGGAGTTCTATGAAAATATGTATAGAGCCCCTACAACATTACAGTCTTGTGGCGTTTGTGTAGTGT GTAATAGTCAAACTATATTGCGCTGTGGTAATTGTATTCGCAAACCATTTTTGTGTTGTAAGTGTTGCTATGACCATGTCATGCACACAGACC ACAAAAATGTTTTGTCTATAAATCCTTACATTTGCTCACAGCCAGGTTGTGGTGAAGCAGATGTTACTAAATTGTACCTCGGAGGTATGTCAT ACTTCTGCGGTAATCATAAACCAAAGTTATCAATACCGTTAGTATCTAATGGTACAGTGTTTGGAATTTACAGGGCTAATTGTGCAGGTAGCG AAAATGTTGATGATTTTAATCAACTAGCTACTACTAATTGGTCTACTGTGGAACCTTATATTTTGGCAAATCGTTGTGTAGATTCGTTGAGAC GCTTTGCTGCAGAGACAGTAAAAGCTACAGAAGAATTACATAAGCAACAATTTGCTAGTGCAGAAGTGAGAGAAGTACTCTCAGATCGTGAAT TGATTCTGTCTTGGGAGCCAGGTAAAACCAGGCCTCCATTGAATAGAAATTATGTTTTCACTGGCTTTCACTTTACTAGAACTAGTAAAGTTC AGCTCGGTGATTTTACATTTGAAAAAGGTGAAGGTAAGGACGTTGTCTATTATCGAGCGACGTCTACTGCTAAATTGTCTGTTGGAGACATTT TTGTTTTAACCTCACACAATGTTGTTTCTCTTATAGCGCCAACGTTGTGTCCTCAGCAAACCTTTTCTAGGTTTGTGAATTTAAGACCTAATG TGATGGTACCTGCGTGTTTTGTAAATAACATTCCATTGTACCATTTAGTAGGCAAGCAGAAGCGTACTACAGTACAAGGCCCTCCTGGCAGTG GTAAATCCCATTTTGCTATAGGATTGGCGGCTTACTTTAGTAACGCCCGTGTCGTTTTTACTGCATGCTCTCATGCAGCTGTTGATGCTTTAT GTGAAAAAGCTTTTAAGTTTCTTAAAGTAGATGATTGCACTCGTATAGTACCTCAAAGGACTACTATCGATTGCTTCTCTAAGTTTAAAGCTA ATGACACAGGCAAAAAGTACATTTTTAGTACTATTAATGCCTTGCCAGAAGTTAGTTGTGACATTCTTTTGGTTGACGAGGTTAGTATGTTGA CCAATTACGAATTGTCTTTTATTAATGGTAAGATAAACTATCAATATGTTGTGTATGTAGGTGATCCTGCTCAATTACCGGCGCCTCGTACGT TGCTTAACGGTTCACTCTCTCCAAAGGATTATAATGTTGTCACAAACCTTATGGTTTGTGTTAAACCTGACATTTTCCTTGCAAAGTGTTACC GTTGTCCTAAAGAAATTGTAGATACTGTTTCTACTCTTGTATATGATGGAAAGTTTATTGCAAATAACCCGGAATCACGTCAGTGTTTCAAGG TTATAGTTAATAATGGTAATTCTGATGTAGGACATGAAAGTGGCTCAGCCTACAACATAACTCAATTAGAATTTGTGAAAGATTTTGTCTGTC GCAATAAGGAATGGCGGGAAGCAACATTCATTTCACCTTATAATGCTATGAACCAGAGAGCCTACCGTATGCTTGGACTTAATGTTCAGACAG TAGACTCGTCTCAAGGTTCGGAGTATGATTATGTTATCTTTTGTGTTACTGCAGATTCGCAGCATGCACTGAATATTAACAGATTCAATGTAG CGCTTACAAGAGCCAAGCGTGGTATACTAGTTGTCATGCGTCAGCGTGATGAACTATATTCAGCTCTTAAGTTTATAGAGCTTGATAGTGTAG CAAGTCTGCAAGGTACAGGCTTGTTTAAAATTTGCAACAAAGAGTTTAGTGGTGTTCACCCAGCTTATGCAGTCACAACTAAGGCTCTTGCTG CAACTTATAAAGTTAATGATGAACTTGCTGCACTTGTTAACGTGGAAGCTGGTTCAGAAATAACATATAAACATCTTATTTCTTTGTTAGGGT TTAAGATGAGTGTTAATGTTGAAGGCTGCCACAACATGTTTATAACACGTGATGAGGCTATCCGCAACGTAAGAGGTTGGGTAGGTTTTGATG TAGAAGCAACACATGCTTGCGGTACTAACATTGGTACTAACCTGCCTTTCCAAGTAGGTTTCTCTACTGGTGCAGACTTTGTAGTTACGCCTG AGGGACTTGTAGATACTTCAATAGGCAATAATTTTGAGCCTGTGAATTCTAAAGCACCTCCAGGTGAACAATTTAATCACTTGAGAGCGTTAT TCAAAAGTGCTAAACCTTGGCATGTTGTAAGGCCAAGGATTGTGCAAATGTTAGCGGATAACCTGTGCAACGTTTCAGATTGTGTAGTGTTTG TCACGTGGTGTCATGGCCTAGAACTAACCACTTTGCGCTATTTTGTTAAAATAGGCAAGGACCAAGTTTGTTCTTGCGGTTCTAGAGCAACAA CTTTTAATTCTCATACTCAGGCTTATGCTTGTTGGAAGCATTGCTTGGGTTTTGATTTTGTTTATAATCCACTCTTAGTGGATATTCAACAGT GGGGTTATTCTGGTAACCTACAATTTAACCATGATTTGCATTGTAATGTGCATGGACACGCACATGTAGCTTCTGCGGATGCTATTATGACGC GTTGTCTTGCAATTAATAATGCATTTTGTCAAGATGTCAACTGGGATTTAACTTACCCTCATATAGCAAATGAGGATGAAGTCAATTCTAGCT GTAGATATTTACAACGCATGTATCTTAATGCATGTGTTGATGCTCTTAAAGTTAACGTTGTCTATGATATAGGCAACCCTAAAGGTATAAAAT GTGTTAGACGTGGAGACTTAAATTTTAGATTCTATGATAAGAATCCAATAGTACCCAATGTCAAGCAGTTTGAGTATGACTATAATCAGCACA AAGATAAGTTTGCTGATGGTCTTTGTATGTTTTGGAATTGTAATGTGGATTGTTATCCCGACAATTCCTTAGTTTGTAGGTACGACACACGAA ATTTGAGTGTGTTTAACCTACCTGGTTGTAATGGTGGTAGCTTGTATGTTAACAAGCATGCATTCCACACACCTAAATTTGATCGCACTAGCT TTCGTAATTTGAAAGCTATGCCATTCTTTTTCTATGACTCATCGCCTTGCGAGACCATTCAATTGGATGGAGTTGCGCAAGACCTTGTGTCAT TAGCTACGAAAGATTGTATCACAAAATGCAACATAGGCGGTGCTGTTTGTAAAAAGCACGCACAAATGTATGCAGATTTTGTGACTTCTTATA ATGCAGCTGTTACTGCTGGTTTTACTTTTTGGGTTACTAATAATTTTAACCCATATAATTTGTGGAAAAGTTTTTCAGCTCTCCAGTCTATCG ACAATATTGCTTATAATATGTATAAGGGTGGTCATTATGATGCTATTGCAGGAGAAATGCCCACTATCGTAACTGGAGATAAAGTTTTTGTTA TAGATCAAGGCGTAGAAAAAGCAGTTTTTTTTAATCAAACAATTCTGCCTACATCTGTAGCGTTTGAGCTGTATGCGAAGAGAAATATTCGCA CACTGCCAAACAACCGTATTTTGAAAGGTTTGGGTGTAGATGTGACTAATGGATTTGTAATTTGGGATTACACGAACCAAACACCACTATACC GTAATACTGTTAAGGTATGTGCATATACAGACATAGAACCAAATGGCCTAATAGTGCTGTATGATGATAGATATGGTGATTACCAGTCTTTTC TAGCTGCTGATAATGCTGTTTTAGTTTCTACACAGTGTTACAAGCGGTATTCGTATGTAGAAATACCGTCAAACCTGCTTGTTCAGAACGGTA TTCCGTTAAAAGATGGAGCGAACCTGTATGTTTATAAGCGTGTTAATGGTGCGTTTGTTACGCTACCTAACACATTAAACACACAGGGTCGCA GTTATGAAACTTTTGAACCTCGTAGTGATGTTGAGCGTGATTTTCTCGACATGTCTGAGGAGAGTTTTGTAGAAAAGTATGGTAAAGAATTAG GTCTACAGCACATACTGTATGGTGAAGTTGATAAGCCCCAATTAGGTGGTTTACACACTGTTATAGGTATGTGCAGACTTTTACGTGCGAATA AGTTGAACGCAAAGTCTGTTACTAATTCTGATTCTGATGTCATGCAAAATTATTTTGTATTGGCAGACAATGGTTCCTACAAGCAAGTGTGTA CTGTTGTGGATTTGCTGCTTGATGATTTCTTAGAACTTCTTAGGAACATACTGAAAGAGTATGGTACTAATAAGTCTAAAGTTGTAACAGTGT CAATTGATTACCATAGCATAAATTTTATGACTTGGTTTGAAGATGGCATTATTAAAACATGTTATCCACAGCTTCAATCAGCATGGACGTGTG GTTATAATATGCCTGAACTTTATAAAGTTCAGAATTGTGTTATGGAACCTTGCAACATTCCTAATTATGGTGTTGGAATAGCGTTGCCAAGTG GTATTATGATGAATGTGGCAAAGTATACACAACTCTGTCAATACCTTTCGAAAACAACAATGTGTGTACCGCATAATATGCGAGTAATGCATT TTGGAGCTGGAAGTGACAAAGGAGTGGCTCCAGGTAGTACTGTTCTTAAACAATGGCTCCCAGAAGGGACACTCCTTGTCGATAATGATATTG TAGACTATGTGTCTGATGCACATGTTTCTGTGCTTTCAGATTGCAATAAATATAAGACAGAGCACAAGTTTGATCTTGTGATATCTGATATGT ATACAGACAATGATTCAAAAAGAAAGCATGAAGGCGTGATAGCCAATAATGGCAATGATGACGTTTTCATATATCTCTCAAGTTTTCTTCGTA ATAATTTGGCTCTAGGTGGTAGTTTTGCTGTAAAAGTGACAGAGACAAGTTGGCACGAAGTTTTATATGACATTGCACAGGATTGTGCATGGT GGACAATGTTTTGTACAGCAGTGAATGCCTCTTCTTCAGAAGCATTCTTGGTTGGTGTTAATTATTTGGGTGCAAGTGAAAAGGTTAAGGTTA GTGGAAAAACGCTGCACGCAAATTATATATTTTGGAGGAATTGTAATTATTTACAAACCTCTGCTTATAGTATATTTGACGTTGCTAAGTTTG ATTTGAGATTGAAAGCAACACCAGTTGTTAATTTGAAAACTGAACAAAAGACAGACTTAGTCTTTAATTTAATTAAGTGTGGTAAGTTACTGG TAAGAGATGTTGGTAACACCTCTTTTACTAGTGACTCTTTTGTGTGTACTATGTAGTGCTGCTTTGTATGACAGTAGTTCTTACGTTTACTAC TACCAAAGTGCCTTTAGACCACCTAATGGTTGGCATTTACACGGGGGTGCTTATGCGGTAGTTAATATTTCTAGCGAATCTAATAATGCAGGC TCTTCACCTGGGTGTATTGTTGGTACTATTCATGGTGGTCGTGTTGTTAATGCTTCTTCTATAGCTATGACGGCACCGTCATCAGGTATGGCT TGGTCTAGCAGTCAGTTTTGTACTGCACACTGTAACTTTTCAGATACTACAGTGTTTGTTACACATTGTTATAAATATGATGGGTGTCCTATA ACTGGCATGCTTCAAAAGAATTTTTTACGTGTTTCTGCTATGAAAAATGGCCAGCTTTTCTATAATTTAACAGTTAGTGTAGCTAAGTACCCT ACTTTTAAATCATTTCAGTGTGTTAATAATTTAACATCCGTATATTTAAATGGTGATCTTGTTTACACCTCTAATGAGACCACAGATGTTACA TCTGCAGGTGTTTATTTTAAAGCTGGTGGACCTATAACTTATAAAGTTATGAGAGAAGTTAAAGCCCTGGCTTATTTTGTTAATGGTACTGCA CAAGATGTTATTTTGTGTGATGGATCACCTAGAGGCTTGTTAGCATGCCAGTATAATACTGGCAATTTTTCAGATGGCTTTTATCCTTTTATT AATAGTAGTTTAGTTAAGCAGAAGTTTATTGTCTATCGTGAAAATAGTGTTAATACTACTTTTACGTTACACAATTTCACTTTTCATAATGAG ACTGGCGCCAACCCTAATCCTAGTGGTGTTCAGAATATTCAAACTTACCAAACACAAACAGCTCAGAGTGGTTATTATAATTTTAATTTTTCC TTTCTGAGTAGTTTTGTTTATAAGGAGTCTAATTTTATGTATGGATCTTATCACCCAAGTTGTAATTTTAGACTAGAAACTATTAATAATGGC TTGTGGTTTAATTCACTTTCAGTTTCAATTGCTTACGGTCCTCTTCAAGGTGGTTGCAAGCAATCTGTCTTTAGTGGTAGAGCAACTTGTTGT TATGCTTATTCATATGGAGGTCCTTCGCTGTGTAAAGGTGTTTATTCAGGTGAGTTAGATCTTAATTTTGAATGTGGACTGTTAGTTTATGTT ACTAAGAGCGGTGGCTCTCGTATACAAACAGCCACTGAACCGCCAGTTATAACTCGACACAATTATAATAATATTACTTTAAATACTTGTGTT GATTATAATATATATGGCAGAACTGGCCAAGGTTTTATTACTAATGTAACCGACTCAGCTGTTAGTTATAATTATCTAGCAGACGCAGGTTTG GCTATTTTAGATACATCTGGTTCCATAGACATCTTTGTTGTACAAGGTGAATATGGTCTTACTTATTATAAGGTTAACCCTTGCGAAGATGTC AACCAGCAGTTTGTAGTTTCTGGTGGTAAATTAGTAGGTATTCTTACTTCACGTAATGAGACTGGTTCTCAGCTTCTTGAGAACCAGTTTTAC ATTAAAATCACTAATGGAACACGTCGTTTTAGACGTTCTATTACTGAAAATGTTGCAAATTGCCCTTATGTTAGTTATGGTAAGTTTTGTATA AAACCTGATGGTTCAATTGCCACAATAGTACCAAAACAATTGGAACAGTTTGTGGCACCTTTACTTAATGTTACTGAAAATGTGCTCATACCT AACAGTTTTAATTTAACTGTTACAGATGAGTACATACAAACGCGTATGGATAAGGTCCAAATTAATTGTCTGCAGTATGTTTGTGGCAATTCT CTGGATTGTAGAGATTTGTTTCAACAATATGGGCCTGTTTGTGACAACATATTGTCTGTAGTAAATAGTATTGGTCAAAAAGAAGATATGGAA CTTTTGAATTTCTATTCTTCTACTAAACCGGCTGGTTTTAATACACCATTTCTTAGTAATGTTAGCACTGGTGAGTTTAATATTTCTCTTCTG TTAACAACTCCTAGTAGTCCTAGAAGGCGTTCTTTTATTGAAGACCTTCTATTTACAAGCGTTGAATCTGTTGGATTACCAACAGATGACGCA TACAAAAATTGCACTGCAGGACCTTTAGGTTTTCTTAAGGACCTTGCGTGTGCTCGTGAATATAATGGTTTGCTTGTGTTGCCTCCCATTATA ACAGCAGAAATGCAAATTTTGTATACTAGTTCTCTAGTAGCTTCTATGGCTTTTGGTGGTATTACTGCAGCTGGTGCTATACCTTTTGCCACA CAACTGCAGGCTAGAATTAATCACTTGGGTATTACCCAGTCACTTTTGTTGAAGAATCAAGAAAAAATTGCTGCTTCCTTTAATAAGGCCATT GGTCGTATGCAGGAAGGTTTTAGAAGTACATCTCTAGCATTACAACAAATTCAAGATGTTGTTAATAAGCAGAGTGCTATTCTTACTGAGACT ATGGCATCACTTAATAAAAATTTTGGTGCTATTTCTTCTATGATTCAAGAAATCTACCAGCAACTTGACGCCATACAAGCAAATGCTCAAGTG GATCGTCTTATAACTGGTAGATTGTCATCACTTTCTGTTTTAGCATCTGCTAAGCAGGCGGAGCATATTAGAGTGTCACAACAGCGTGAGTTA GCTACTCAGAAAATTAATGAGTGTGTTAAGTCACAGTCTATTAGGTACTCCTTTTGTGGTAATGGACGACATGTTCTAACCATACCGCAAAAT GCACCTAATGGTATAGTGTTTATACACTTTTCTTATACTCCAGATAGTTTTGTTAATGTTACTGCAATAGTGGGTTTTTGTGTAAAGCCAGCT AATGCTAGTCAGTATGCAATAGTACCCGCTAATGGTAGGGGTATTTTTATACAAGTTAATGGTAGTTACTACATCACAGCACGAGATATGTAT ATGCCAAGAGCTATTACTGCAGGAGATATAGTTACGCTTACTTCTTGTCAAGCAAATTATGTAAGTGTAAATAAGACCGTCATTACTACATTC GTAGACAATGATGATTTTGATTTTAATGACGAATTGTCAAAATGGTGGAATGACACTAAGCATGAGCTACCAGACTTTGACAAATTCAATTAC ACAGTACCTATACTTGACATTGATAGTGAAATTGATCGTATTCAAGGCGTTATACAGGGTCTTAATGACTCTTTAATAGACCTTGAAAAACTT TCAATACTCAAAACTTATATTAAGTGGCCTTGGTATGTGTGGTTAGCCATAGCTTTTGCCACTATTATCTTCATCTTAATACTAGGATGGGTT TTCTTCATGACTGGATGTTGTGGTTGTTGTTGTGGATGCTTTGGCATTATGCCTCTAATGAGTAAGTGTGGTAAGAAATCTTCTTATTACACG ACTTTTGATAACGATGTGGTAACTTAACAATACAGACCTAAAAAGTCTGTTTAATGATTCAAAGTCCCACGTCCTTCCTAATAGTATTAATTT TTCTTTGGTGTAAACTTGTACTAAGTTGTTTTAGAGAGTTTATTATAGCGCTCCAACAACTAATACAAGTTTTACTCCAAATTATCAATAGTA ACTTACAGCCTAGACTGACCCTTTGTCACAGTCTAGACTAATGTTAAACTTAGAAGCAATTATTGAAACTGGTGAGCAAGTGATTCAAAAAAT CAGTTTCAATTTACAGCATATTTCAAGTGTATTAAACACAGAAGTATTTGACCCCTTTGACTATTGTTATTACAGAGGAGGTAATTTTTGGGA AATAGAGTCAGCTGAAGATTGTTCAGGTGATGATGAATTTATTGAATAAGTCGCTAGAGGAAAATGGAAGTTTTCTAACAGCGCTTTATATAT TTGTAGGATTTTTAGCACTTTATCTTCTAGGTAGAGCACTTCAAGCATTTGTACAGGCTGCTGATGCTTGTTGTTTATTTTGGTATACATGGG TAGTAATTCCAGGAGCTAAGGGTACAGCCTTTGTATATAAGTATACATATGGTAGAAAACTTAACAATCCGGAATTAGAAGCAGTTATTGTCA ACGAGTTTCCTAAGAACGGTTGGAATAATAAAAATCCAGCAAATTTTCAAGATGTCCAACGAGACAAATTGTACTCTTGACTTTGAACAGTCA GTTGAGCTTTTTAAAGAGTATAATTTATTTATAACTGCATTCTTGTTGTTCTTAACCATAATACTTCAGTATGGCTATGCAACAAGAAGTAAG TTTATTTATATACTGAAAATGATAGTGTTATGGTGCTTTTGGCCCCTTAACATTGCAGTAGGTGTAATTTCATGTATATACCCACCAAACACA GGAGGTCTTGTCGCAGCGATAATACTTACAGTGTTTGCGTGTCTGTCTTTTGTAGGTTATTGGATCCAGAGTATTAGACTCTTTAAGCGGTGT AGGTCATGGTGGTCATTTAACCCAGAATCTAATGCCGTAGGTTCAATACTCCTAACTAATGGTCAACAATGTAATTTTGCTATAGAGAGTGTG CCAATGGTGCTTTCTCCAATTATAAAGAATGGTGTTCTTTATTGTGAGGGTCAGTGGCTTGCTAAGTGTGAACCAGACCACTTGCCTAAAGAT ATATTTGTTTGTACACCGGATAGACGTAATATCTACCGTATGGTGCAGAAATATACTGGTGACCAAAGCGGAAATAAGAAACGGTTTGCTACG TTTGTCTATGCAAAGCAGTCAGTAGATACTGGCGAGCTAGAAAGTGTAGCAACAGGAGGGAGTAGTCTTTACACCTAAATGTGTGTGTGTAGA GAGTATTTAAAATTATTCTTTAATAGTGCCTCTATTTTAAGAGCGCATAATAGTATTATTTTTGAGGATATTAATATAAATCCTCTCTGTTTT ATACTCTCTTTTCAAGAGCTATTATTTAAAAAACAGTTTTTCCACTCTTTTGTGCCAAAAACTATTGTTGTTAATGGTGTAACCTTTCAAGTA GATAATGGAAAAGTCTACTACGAAGGAAAACCAATTTTTCAGAAAGGTTGTTGTAGGTTGTGGTTGAGTTATAAAAAAGATTAAACTACCTAC TACACTTATTTTTATAAGAGGCGTTTTATCTTACAAGCGCTTAATAAATACGGACGATGAAATGGCTGACTAGTTTTGTAAGGGCAGTTATTT CATGTTATAAACCCCTATTATTAACTCAATTAAGAGTATTAGATAGGTTAATCTTAGATCATGGACCAAAACACATCTTAACGTGTGTTAGGT GCGTGATTTTGTTTCAATTAGATTTAGTTTATAGGTTGGCGTATACGCCTACTCAATCGCTGGTATGAATAATAGTAAAGATAATCCTTTTTG CGGAGCAATAGCAAGAAAAGCGCGAATTTATCTGAGAGAAGGATTAGATTGTGTTTACTTTCTTAACAAAGCAGGACAAGCAGAGTCTTGTCC CGCGTGTACCTCTCTAGTATTCCAGGGGAAAACTTGTGAGGAACACAAATATAATAATAATCTTTTGTCATGGCAAGCGGTAAGGCAACTGGA AAGACAGATGCCCCAGCTCCAGTCATCAAACTAGGAGGACCAAAGCCACCTAAAGTTGGTTCTTCTGGAAATGTATCTTGGTTTCAAGCAATA AAAGCCAAGAAGTTAAATTCACCTCCGCCTAAGTTTGAAGGTAGCGGTGTTCCTGATAATGAAAATCTAAAACCAAGTCAGCAGCATGGATAT TGGAGACGCCAAGCTAGGTTTAAGCCAGGTAAAGGTGGAAGAAAACCAGTCCCAGATGCTTGGTATTTTTACTATACTGGAACAGGACCAGCC GCTAACCTGAATTGGGGTGATAGCCAAGATGGTATAGTGTGGGTTGCTGGTAAGGGTGCTGATACTAAATTTAGATCTAATCAGGGTACTCGT GACTCTGACAAGTTTGACCAATATCCGCTACGGTTTTCAGACGGAGGACCTGATGGTAATTTCCGTTGGGATTTCATTCCTCTGAATCGTGGC AGGAGTGGGAGATCAACAGCAGCTTCATCAGCAGCATCTAGTAGAGCACCATCACGTGAAGTTTCGCGTGGTCGCAGGAGTGGTTCTGAAGAT GATCTTATTGCTCGTGCAGCAAGGATAATTCAGGATCAGCAGAAGAAGGGTTCTCGCATTACAAAGGCTAAGGCTGATGAAATGGCTCACCGC CGGTATTGCAAGCGCACTATTCCACCTAATTATAAGGTTGATCAAGTGTTTGGTCCCCGTACTAAAGGTAAGGAGGGAAATTTTGGTGATGAC AAGATGAATGAGGAAGGTATTAAGGATGGGCGCGTTACAGCAATGCTCAACCTAGTTCCTAGCAGCCATGCTTGTCTTTTCGGAAGTAGAGTG ACGCCCAGACTTCAACCAGATGGGCTGCACTTGAAATTTGAATTTACTACTGTGGTCCCACGTGATGATCCGCAGTTTGATAATTATGTAAAA ATTTGTGATCAGTGTGTTGATGGTGTAGGAACACGTCCAAAAGATGATGAACCAAGACCAAAGTCACGCTCAAGTTCAAGACCTGCAACAAGA GGAAATTCTCCAGCGCCAAGACAGCAGCGCCCTAAGAAGGAGAAAAAGCCAAAGAAGCAGGATGATGAAGTGGATAAAGCATTGACCTCAGAT GAGGAGAGGAACAATGCACAGCTGGAATTTGATGATGAACCCAAGGTAATTAACTGGGGGGATTCAGCCCTAGGAGAGAATGAACTTTGAGTA AAATTCAATAGTAAGAGTTAAGGAAGATAGGCATGTAGCTTGATTACCTACATGTCTATCGCCAGGGAAATGTCTAATTTGTCTACTTAGTAG CCTGGAAACGAACGGTAGACCCTTAGATTTTAATTTAGTTTAATTTTTAGTTTAGTTTAAGTTAGTTTAGAGTAGGTATAAAGATGCCAGTGC CGGGGCCACGCGGAGTACGACCGAGGGTACAGCACTAGGACGCCCATTAGGGGAAGAGCTAAATTTTAGTTTAAGTTAAGTTTAATTGGCTAT GTATAGTTAAAATTTATAGGCTAGTATAGAGTTAGAGCAAAAAAAAAAAAAAAAAAAAAAAAAAA

Accessory Proteins

All coronaviruses 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 nucleotide sequence for gene 3 and amino acid sequences for accessory proteins 3a and 3b in IBV MK41 are provided as SEQ ID NOs: 2-4, respectively.

(gene 3 nucleotide sequence-nucleotides 23831 to 24539 of SEQ ID NO: 1) SEQ ID NO: 2 CTTAACAATACAGACCTAAAAAGTCTGTTTAATGATTCAAAGTCCCACGT CCTTCCTAATAGTATTAATTTTTCTTTGGTGTAAACTTGTACTAAGTTGT TTTAGAGAGTTTATTATAGCGCTCCAACAACTAATACAAGTTTTACTCCA AATTATCAATAGTAACTTACAGCCTAGACTGACCCTTTGTCACAGTCTAG ACTAATGTTAAACTTAGAAGCAATTATTGAAACTGGTGAGCAAGTGATTC AAAAAATCAGTTTCAATTTACAGCATATTTCAAGTGTATTAAACACAGAA GTATTTGACCCCTTTGACTATTGTTATTACAGAGGAGGTAATTTTTGGGA AATAGAGTCAGCTGAAGATTGTTCAGGTGATGATGAATTTATTGAATAAG TCGCTAGAGGAAAATGGAAGTTTTCTAACAGCGCTTTATATATTTGTAGG ATTTTTAGCACTTTATCTTCTAGGTAGAGCACTTCAAGCATTTGTACAGG CTGCTGATGCTTGTTGTTTATTTTGGTATACATGGGTAGTAATTCCAGGA GCTAAGGGTACAGCCTTTGTATATAAGTATACATATGGTAGAAAACTTAA CAATCCGGAATTAGAAGCAGTTATTGTCAACGAGTTTCCTAAGAACGGTT GGAATAATAAAAATCCAGCAAATTTTCAAGATGTCCAACGAGACAAATTG TACTCTTGA (accessory protein 3a amino acid sequence) SEQ ID NO: 3 MIQSPTSFLIVLIFLWCKLVLSCFREFIIALQQLIQVLLQIINSNLQPRL TLCHSLD (accessory protein 3b amino acid sequence) SEQ ID NO: 4 MLNLEAIIETGEQVIQKISFNLQHISSVLNTEVFDPFDYCYYRGGNFWEI ESAEDCSGDDEFIE

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

Nsp-3 is a large protein consisting of numerous distinct domains including a ubiquitin-like domain, an acidic domain, a papain-like protease (PL pro) domain, multiple transmembrane domains, and a macrodomain, which are separated by disordered linkers. The macrodomain is a globular domain with central sheets flanked by helices located at the N terminus of nsp-3. It contains ADP-ribose-1-phosphatase (ADRP) activity and binds to various forms of ADP-ribose. This domain is highly conserved in coronaviruses but found in only a few other virus families.

The nucleotide and protein sequences for nsp-3 in IBV M41 are provided as SEQ ID NOs: 5 and 6, respectively.

(nsp-3 nucleotide sequence-nucleotides 2548-7329 of SEQ ID NO: 1) SEQ ID NO: 5 GGTAAAACTGTCACCTTTGGAGAAACTACTGTGCAAGAAATACCACCACCTGATGTTGTGTTTATT AAGGTTAGCATTGAGTGTTGTGGTGAACCATGGAATACAATCTTCAAAAAGGCTTATAAGGAGCCC ATTGAAGTAGAGACAGACCTCACAGTTGAACAATTGCTCTCTGTGGTCTATGAGAAAATGTGTGAT GATCTCAAGCTGTTTCCGGAGGCTCCAGAACCACCACCATTTGAGAATGTCACACTTGTTGATAAG AATGGTAAAGATTTGGATTGCATAAAATCATGCCATCTGATCTATCGTGATTATGAGAGCGATGAT GACATCGAGGAAGAAGATGCAGAAGAATGTGACACGGATTCAGGTGATGCTGAGGAGTGTGACACT AATTCAGAATGTGAAGAAGAAGATGAGGATACTAAAGTGTTGGCTCTTATACAAGACCCGGCAAGT AACAAATATCCTCTGCCTCTTGATGATGATTATAGCGTCTACAATGGATGTATTGTTCATAAGGAC GCTCTCGATGTTGTGAATTTACCATCTGGTGAAGAAACCTTTGTTGTCAATAACTGCTTTGAAGGG GCTGTTAAAGCTCTTCCGCAGAAAGTTATTGATGTTCTAGGTGACTGGGGTGAGGCTGTTGATGCG CAAGAACAATTGTGTCAACAAGAATCAACTCGGGTCATATCTGAGAAATCAGTTGAGGGTTTTACT GGTAGTTGTGATGCAATGGCTGAACAAGCTATTGTTGAAGAGCAGGAAATAGTACCTGTTGTTGAA CAAAGTCAGGATGTAGTTGTTTTTACACCTGCAGACCTAGAAGTTGTTAAAGAAACAGCAGAAGAG GTTGATGAGTTTATTCTCATTTCTGCTGTCCCTAAAGAAGAAGTTGTGTCTCAGGAGAAAGAGGAG CCACAGGTTGAGCAAGAGCCTACCCTAGTTGTTAAAGCACAACGTGAGAAGAAGGCTAAAAAGTTC AAAGTTAAACCAGCTACATGTGAAAAACCCAAATTTTTGGAGTACAAAACATGTGTGGGTGATTTG GCTGTTGTAATTGCCAAAGCATTGGATGAGTTTAAAGAGTTCTGCATTGTAAACGCTGCAAATGAG CACATGTCGCATGGTGGTGGCGTTGCAAAGGCAATTGCAGACTTTTGTGGACCGGACTTTGTTGAA TATTGCGCGGACTATGTTAAGAAACATGGTCCACAGCAAAAACTTGTCACACCTTCATTTGTTAAA GGCATTCAATGTGTGAATAATGTTGTAGGACCTCGCCATGGAGACAGCAACTTGCGTGAGAAGCTT GTTGCTGCTTACAAGAGTGTTCTTGTAGGTGGAGTGGTTAACTATGTTGTGCCAGTTCTCTCATCA GGGATTTTTGGTGTAGATTTTAAAATATCAATAGATGCTATGCGCGAAGCTTTTAAAGGTTGTGCC ATACGCGTTCTTTTATTTTCTCTGAGTCAAGAACACATCGATTATTTCGATGCAACTTGTAAGCAG AAGACAATTTATCTTACGGAGGATGGTGTTAAATACCGCTCTGTTGTTTTAAAACCTGGTGATTCT TTGGGTCAATTTGGACAGGTTTTTGCAAGAAATAAGGTAGTCTTTTCGGCTGATGATGTTGAGGAT AAAGAAATCCTCTTTATACCCACAACTGACAAGACTATTCTTGAATATTATGGTTTAGATGCGCAA AAGTATGTAACATATTTGCAAACGCTTGCGCAGAAATGGGATGTTCAATATAGAGACAATTTTGTT ATATTAGAGTGGCGTGACGGAAATTGCTGGATTAGTTCAGCAATAGTTCTCCTTCAAGCTGCTAAA ATTAGATTTAAAGGTTTTCTTGCAGAAGCATGGGCTAAACTGTTGGGTGGAGATCCTACAGACTTT GTTGCCTGGTGTTATGCAAGTTGCAATGCTAAAGTAGGTGATTTTTCAGATGCTAATTGGCTTTTG GCCAATTTAGCAGAACATTTTGACGCAGATTACACAAATGCACTTCTTAAGAAGTGTGTGTCGTGC AATTGTGGTGTTAAGAGTTATGAACTTAGGGGTCTTGAAGCCTGTATTCAGCCAGTTCGAGCACCT AATCTTCTACATTTTAAAACGCAATATTCAAATTGCCCAACCTGTGGTGCAAGTAGTACGGATGAA GTAATAGAAGCTTCATTACCGTACTTATTGCTTTTTGCTACTGATGGTCCTGCTACAGTTGATTGT GATGAAAATGCTGTAGGGACTGTTGTTTTCATTGGCTCTACTAATAGTGGCCATTGTTATACACAA GCCGATGGTAAGGCTTTTGACAATCTTGCTAAGGATAGAAAATTTGGAAGGAAGTCGCCTTACATT ACAGCAATGTATACACGTTTTTCTCTTAGGAGTGAAAATCCCCTACTTGTTGTTGAACATAGTAAG GGTAAAGCTAAAGTAGTAAAAGAAGATGTTTCTAACCTTGCTACTAGTTCTAAAGCCAGTTTTGAC GATCTTACTGACTTTGAACAGTGGTATGATAGCAACATCTATGAGAGTCTTAAAGTGCAGGAGACA CCTGATAATCTTGATGAATATGTGTCATTTACGACAAAGGAAGATTCTAAGTTGCCACTGACACTT AAAGTTAGAGGTATCAAATCAGTTGTTGACTTTAGGTCTAAGGATGGTTTTACTTATAAGTTAACA CCTGATACTGATGAAAATTCAAAAACACCAGTCTACTACCCAGTCTTGGATTCTATTAGTCTTAGG GCAATATGGGTTGAAGGCAGTGCTAATTTTGTTGTTGGGCATCCAAATTATTATAGTAAGTCTCTC CGAATTCCCACGTTTTGGGAAAATGCCGAGAGCTTTGTTAAAATGGGTTATAAAATTGATGGTGTA ACTATGGGCCTTTGGCGTGCAGAACACCTTAATAAACCTAATTTGGAGAGAATTTTTAACATTGCT AAGAAAGCTATTGTTGGATCTAGTGTTGTTACTACGCAGTGTGGTAAAATACTAGTTAAAGCAGCT ACATACGTTGCCGATAAAGTAGGTGATGGTGTAGTTCGCAATATTACAGATAGAATTAAGGGTCTT TGTGGATTCACACGTGGCCATTTTGAAAAGAAAATGTCCCTACAATTTCTAAAGACACTTGTGTTC TTTTTCTTTTATTTCTTAAAGGCTAGTGCTAAGAGTTTAGTTTCTAGCTATAAGATTGTGTTATGT AAGGTGGTGTTTGCTACCTTACTTATAGTGTGGTTTATATACACAAGTAATCCAGTAGTGTTTACT GGAATACGTGTGCTAGACTTCCTATTTGAAGGTTCTTTATGTGGTCCTTATAATGACTACGGTAAA GATTCTTTTGATGTGTTACGCTATTGTGCAGGTGATTTTACTTGTCGTGTGTGTTTACATGATAGA GATTCACTTCATCTGTACAAACATGCTTATAGCGTAGAACAAATTTATAAGGATGCAGCTTCTGGC ATTAACTTTAATTGGAATTGGCTTTATTTGGTCTTTCTAATATTATTTGTTAAGCCAGTGGCAGGT TTTGTTATTATTTGTTATTGTGTTAAGTATTTGGTATTGAGTTCAACTGTGTTGCAAACTGGTGTA GGTTTTCTAGATTGGTTTGTAAAAACAGTTTTTACCCATTTTAATTTTATGGGAGCGGGATTTTAT TTCTGGCTCTTTTACAAGATATACGTACAAGTGCATCATATATTGTACTGTAAGGATGTAACATGT GAAGTGTGCAAGAGAGTTGCACGCAGCAACAGGCAAGAGGTTAGCGTTGTAGTTGGTGGACGCAAG CAAATAGTGCATGTTTACACTAATTCTGGCTATAACTTTTGTAAGAGACATAATTGGTATTGTAGA AATTGTGATGATTATGGTCACCAAAATACATTTATGTCCCCTGAAGTTGCTGGCGAGCTTTCTGAA AAGCTTAAGCGCCATGTTAAACCTACAGCATATGCTTACCACGTTGTGTATGAGGCATGCGTGGTT GATGATTTTGTTAATTTAAAATATAAGGCTGCAATTCCTGGTAAGGATAATGCATCTTCTGCTGTT AAGTGTTTCAGTGTTACAGATTTTTTAAAGAAAGCTGTTTTTCTTAAGGAGGCATTGAAATGTGAA CAAATATCTAATGATGGTTTTATAGTGTGTAATACACAGAGTGCGCATGCACTAGAGGAAGCAAAG AATGCAGCCGTCTATTATGCGCAATATCTGTGTAAGCCAATACTTATACTTGACCAGGCACTTTAT GAGCAATTAATAGTAGAGCCTGTGTCTAAGAGTGTTATAGATAAAGTGTGTAGCATTTTGTCTAAT ATAATATCTGTAGATACTGCAGCTTTAAATTATAAGGCAGGCACACTTCGTGATGCTCTGCTTTCT ATTACTAAAGACGAAGAAGCCGTAGATATGGCTATCTTCTGCCACAATCATGAAGTGGAATACACT GGTGACGGTTTTACTAATGTGATACCGTCATATGGTATGGACACTGATAAGTTGACACCTCGTGAT AGAGGGTTTTTGATAAATGCAGATGCTTCTATTGCTAATTTAAGAGTCAAAAATGCTCCTCCGGTA GTATGGAAGTTTTCTGATCTTATTAAATTGTCTGACAGTTGCCTTAAATATTTAATTTCAGCTACT GTCAAGTCAGGAGGTCGTTTCTTTATAACAAAGTCTGGTGCTAAACAAGTTATTTCTTGTCATACC CAGAAACTGTTGGTAGAGAAAAAGGCAGGT (nsp-3 amino acid sequence) SEQ ID NO: 6 GKTVTFGETTVQEIPPPDVVFIKVSIECCGEPWNTIFKKAYKEPIEVETDLTVEQLLSVVYEKMCD DLKLFPEAPEPPPFENVTLVDKNGKDLDCIKSCHLIYRDYESDDDIEEEDAEECDTDSGDAEECDT NSECEEEDEDTKVLALIQDPASNKYPLPLDDDYSVYNGCIVHKDALDVVNLPSGEETFVVNNCFEG AVKALPQKVIDVLGDWGEAVDAQEQLCQQESTRVISEKSVEGFTGSCDAMAEQAIVEEQEIVPVVE QSQDVVVFTPADLEVVKETAEEVDEFILISAVPKEEVVSQEKEEPQVEQEPTLVVKAQREKKAKKF KVKPATCEKPKFLEYKTCVGDLAVVIAKALDEFKEFCIVNAANEHMSHGGGVAKAIADFCGPDFVE YCADYVKKHGPQQKLVTPSFVKGIQCVNNVVGPRHGDSNLREKLVAAYKSVLVGGVVNYVVPVLSS GIFGVDFKISIDAMREAFKGCAIRVLLFSLSQEHIDYFDATCKQKTIYLTEDGVKYRSVVLKPGDS LGQFGQVFARNKVVFSADDVEDKEILFIPTTDKTILEYYGLDAQKYVTYLQTLAQKWDVQYRDNFV ILEWRDGNCWISSAIVLLQAAKIRFKGFLAEAWAKLLGGDPTDFVAWCYASCNAKVGDFSDANWLL ANLAEHFDADYTNALLKKCVSCNCGVKSYELRGLEACIQPVRAPNLLHFKTQYSNCPTCGASSTDE VIEASLPYLLLFATDGPATVDCDENAVGTVVFIGSTNSGHCYTQADGKAFDNLAKDRKFGRKSPYI TAMYTRFSLRSENPLLVVEHSKGKAKVVKEDVSNLATSSKASFDDLTDFEQWYDSNIYESLKVQET PDNLDEYVSFTTKEDSKLPLTLKVRGIKSVVDFRSKDGFTYKLTPDTDENSKTPVYYPVLDSISLR AIWVEGSANFVVGHPNYYSKSLRIPTFWENAESFVKMGYKIDGVTMGLWRAEHLNKPNLERIFNIA KKAIVGSSVVTTQCGKILVKAATYVADKVGDGVVRNITDRIKGLCGFTRGHFEKKMSLQFLKTLVF FFFYFLKASAKSLVSSYKIVLCKVVFATLLIVWFIYTSNPVVFTGIRVLDFLFEGSLCGPYNDYGK DSFDVLRYCAGDFTCRVCLHDRDSLHLYKHAYSVEQIYKDAASGINFNWNWLYLVFLILFVKPVAG FVIICYCVKYLVLSSTVLQTGVGFLDWFVKTVFTHFNFMGAGFYFWLFYKIYVQVHHILYCKDVTC EVCKRVARSNRQEVSVVVGGRKQIVHVYTNSGYNFCKRHNWYCRNCDDYGHQNTFMSPEVAGELSE KLKRHVKPTAYAYHVVYEACVVDDFVNLKYKAAIPGKDNASSAVKCFSVTDFLKKAVFLKEALKCE QISNDGFIVCNTQSAHALEEAKNAAVYYAQYLCKPILILDQALYEQLIVEPVSKSVIDKVCSILSN IISVDTAALNYKAGTLRDALLSITKDEEAVDMAIFCHNHEVEYTGDGFTNVIPSYGMDTDKLTPRD RGFLINADASIANLRVKNAPPVVWKFSDLIKLSDSCLKYLISATVKSGGRFFITKSGAKQVISCHT QKLLVEKKAG

Reduced Pathogenicity

The live, attenuated coronavirus of the present invention comprises at least one mutation which causes the virus to have reduced pathogenicity compared to the corresponding wild-type coronavirus.

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 wheezing, 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.

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, wheezing, snicking, depression, ruffled feathers and loss of tracheal ciliary activity.

The attenuated coronavirus of the present invention may cause a reduced level of clinical symptoms compared to the corresponding wild-type coronavirus.

For example an attenuated coronavirus 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 wild type virus.

An attenuated coronavirus 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 a wild type virus.

An attenuated coronavirus 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.

An attenuated coronavirus according to the present invention may cause clinical symptoms, as defined in Table 2, at a lower level than a wild type coronavirus.

TABLE 2 IBV severity limits based on clinical signs: Snicking (sneezing) Nasal exudate IBV specific: Mild (N.B. Respiratory signs Watery eyes become apparent from 2-3 dpi if they Swollen infraorbital sinuses {open oversize bracket} are going to occur and can continue for Rales (vibration in trachea or bronchi region) up to 7 d). Hunched posture/depressed Fluffed up feathers {open oversize bracket} Mild, if exceed 2 d increase to Eating and drinking less moderate Drinking in excess: evident IBV specific: Mild, if exceed 24 h increase to by fluid filled crop or moderate for a max of 2 d. If still drinking in excess measured water intake then kill by schedule 1 method. Less active but still evade capture Weight loss {open oversize bracket} Mild, if exceed 1 d increase to Not eating or drinking moderate. Birds sit alone and does not evade capture Severe respiratory distress: e.g. excessive gasping {open oversize bracket} Moderate: birds at end point. Kill by schedule 1 Snicking and/or rales for 7 d in total method. Severe: report to project license Found dead holder. Full post-mortem to be performed.

The attenuated coronavirus of the present invention may 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 attenuated 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 attenuated coronavirus 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 attenuated coronavirus propagates 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 attenuated coronavirus demonstrates 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 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 Bestfit 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

Ammayappan et al (Arch Virol (2009) 154:495-499) 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 coronavirus of the present invention comprises a mutation in non-structural protein nsp-3 and/or deletion of accessory proteins 3a and/or 3b, which mutation causes the virus to have reduced pathogenicity compared to a wild-type coronavirus.

The mutation in nsp-3 may comprise a mutation in the adenosine diphosphate-ribose-1′-phophatase (ADRP) region of nsp-3. The ADRP region of nsp-3 may comprise or consist of residues 332 to 491 of SEQ ID NO: 6 (encoded by nucleotides 994 to 1473 of SEQ ID NO: 5, for example). Accordingly, the mutation in nsp-3 may comprise one or more amino acids mutations compared to the amino acids shown as positions 332 to 491 of SEQ ID NO: 6. The mutation in nsp-3 may comprise one or more amino acid mutations compared to the amino acids shown as positions 332 to 491, 350 to 470, 350 to 450, 350 to 425, 350 to 400, 350 to 380, 360 to 380 or 370 to 380 of SEQ ID NO: 6. The mutation in nsp-3 may comprise one or more amino acid mutations compared to the amino acids shown as positions 370 to 380 of SEQ ID NO: 6.

The mutation in nsp-3 may comprise one or more amino acid mutations selected from the list of:

    • a) Asn (N) to Ala (A) at position 373 in SEQ ID NO: 6; and
    • b) Gly (G) to Ser (S) at position 379 in SEQ ID NO: 6.

The mutation in nsp-3 may comprise one or more nucleotide substitutions which encodes an amino acid mutation as described herein.

The mutation in nsp-3 may comprise one or more nucleotide substitutions selected from the list of:

    • a) A to G at nucleotide position 1116 and A to C at nucleotide position 1117 compared to the sequence shown as SEQ ID NO: 5; and
    • b) G to A at nucleotide position 1138 compared to the sequence shown as SEQ ID NO: 5.

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 mutation in nsp-3 may comprise the amino acid mutation Asn (N) to Ala (A) at position 373 in SEQ ID NO: 3.

The mutation in nsp-3 may comprise the amino acid mutation Gly (G) to Ser (S) at position 379 in SEQ ID NO: 3.

The mutation in nsp-3 may comprise the amino acid mutations Asn (N) to Ala (A) at position 373 and Gly (G) to Ser (S) at position 379 in SEQ ID NO: 3.

The attenuated coronavirus may also be defined at the nucleotide level.

For example the nucleotide sequence of the attenuated coronavirus of the present invention may comprise one or more mutations selected from the list of:

    • a) A to G at nucleotide position 1116 and A to C at nucleotide position 1117 compared to the sequence shown as SEQ ID NO: 5; and
    • b) G to A at nucleotide position 1138 compared to the sequence shown as SEQ ID NO: 5.

The mutation in nsp-3 may comprise the mutation A to G at nucleotide position 1116 and A to C at nucleotide position 1117 compared to the sequence shown as SEQ ID NO: 5.

The mutation in nsp-3 may comprise the mutation G to A at nucleotide position 1138 compared to the sequence shown as SEQ ID NO: 5.

The mutation in nsp-3 may comprise the mutation A to G at nucleotide position 1116, A to C at nucleotide position 1117 and G to A at nucleotide position 1138 compared to the sequence shown as SEQ ID NO: 5.

The live, attenuated coronavirus of the present invention may comprise deletion of accessory proteins 3a and/or 3b. This mutation may be achieved by partial or full deletion of gene 3. Therefore in one aspect of the invention all or a portion of accessory protein 3a is deleted. According to another aspect of the invention all or a portion of accessory protein 3b is deleted. In a further aspect of the invention all or a portion of both accessory proteins 3a and 3b are deleted. In one aspect of the invention accessory protein 3a is deleted. According to another aspect of the invention accessory protein 3b is deleted. In a further aspect of the invention both accessory proteins 3a and 3b are deleted.

The deletion of accessory proteins 3a and 3b may comprise a deletion from about nucleotide position 37 to about 384 of the gene 3 sequence shown as SEQ ID NO: 2. The deletion of accessory proteins 3a and 3b may comprise a deletion from nucleotide position 37 to 384 of the gene 3 sequence shown as SEQ ID NO: 2.

The deletion of accessory proteins 3a and 3b may comprise a deletion from about nucleotide position 23867 to about 24214 of the IBV M41-CK sequence shown as SEQ ID NO: 1. The deletion of accessory proteins 3a and 3b may comprise a deletion from nucleotide position 23867 to 24214 of the IBV M41-CK sequence shown as SEQ ID NO:

The nucleotide sequence may comprise any combination of the nucleotide mutations listed above.

In one embodiment, the coronavirus of the present invention may comprise a mutation in non-structural protein nsp-3 and deletion of accessory proteins 3a and/or 3b.

In a further embodiment, the coronavirus of the present invention may comprise a mutation in the ADRP region of nsp-3 and deletion of accessory proteins 3a and 3b.

For example the nucleotide sequence of the attenuated coronavirus of the present invention may comprise the following mutations:

    • a) A to G at nucleotide position 1116 and A to C at nucleotide position 1117 compared to the sequence shown as SEQ ID NO: 5; and/or
    • b) G to A at nucleotide position 1138 compared to the sequence shown as SEQ ID NO: 5; and
    • c) deletion from nucleotide position 37 to 384 of the gene 3 sequence shown as SEQ ID NO: 2.

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 attenuated coronavirus. 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.

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 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 nsp-3 gene or gene 3;
    • (iii) allowing homologous recombination to occur between the gene sequences in the plasmid and the corresponding sequences in the recombining virus genome to produce a modified gene;
    • (iv) selecting for recombining virus comprising the modified gene.

The term ‘modified gene’ refers to a nsp-3 gene or gene 3 which comprises a mutation 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 gene but comprises a nucleotide sequence which causes it to encode a variant protein as defined herein, or in the case of gene 3, to not encode part or all of the proteins 3a and 3b

The recombination may involve all or part of the modified gene. 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 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 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 gene by, for example, PCR and sequencing.

Large stocks of the recombining virus including the modified 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 a modified gene of the present invention.

The cell may be able to produce recombinant recombining virus (e.g. vaccinia virus) containing the mutation(s).

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 be 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-HeI, carbonic acid, tartaric acid, succinic acid, acetic acid, or phthalic acid, Tris, 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 with 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 modified 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 or 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.

Definitions of terms appear throughout the specification. Before the exemplary embodiments are described in more detail, it is to be understood that this disclosure is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Singleton, et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY, 20 ED., John Wiley and Sons, New York (1994), and Hale & Marham, THE HARPER COLLINS DICTIONARY OF BIOLOGY, Harper Perennial, NY (1991) provide one of skill with a general dictionary of many of the terms used in this disclosure.

This disclosure is not limited by the exemplary methods and materials disclosed herein, and any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of this disclosure. Numeric ranges are inclusive of the numbers defining the range. Unless otherwise indicated, any nucleic acid sequences are written left to right in 5′ to 3′ orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively.

Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within this disclosure. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither or both limits are included in the smaller ranges is also encompassed within this disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in this disclosure.

It must be noted that as used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise.

Thus, for example, reference to “an enzyme” includes a plurality of such candidate agents and equivalents thereof known to those skilled in the art, and so forth.

The terms “comprising”, “comprises” and “comprised of” as used herein are synonymous with “including”, “includes” or “containing”, “contains”, and are inclusive or open-ended and do not exclude additional, non-recited members, elements or method steps. The terms “comprising”, “comprises” and “comprised of” also include the term “consisting of”.

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 Struct 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 (TDS) system (see PCT/GB2010/001293).

Example 2—Determining the Pathogenicity of Recombinant M41 Viruses

Three recombinants were produced using the reverse genetics system described in Example 1. These recombinants were named rIBV M41K-S-ADRP, rIBV M41K-A-ADRP and rIBV M41K-del3ab.

The three recombinants were shown to grow in a similar manner in chicken kidney cells as wild-type M41-CK/M41-K (FIGS. 7 and 8).

rIBV M41K-S-ADRP has a mutation at nucleotide position G3685A in the adenosine diphosphate-ribose-1′-phosphatase (ADRP) region of nsp-3 resulting in an amino acid mutation from G to S. This mutation was introduced into the rVV containing M41-K using the plasmid shown in FIG. 1.

rIBV M41K-A-ADRP has two mutations at nucleotide positions A3664G and A3665C in the ADRP region of nsp-3 resulting in an amino acid mutation from N to A. This mutation was introduced into the rVV containing M41-K using the plasmid shown in FIG. 2.

rIBV M41K-del3ab has a deletion from nucleotide position 23867 to 24214 in gene 3 resulting in a lack of accessory proteins 3a and 3b. This region was deleted from the rVV containing M41-K using the plasmid shown in FIG. 3.

The three recombinant viruses 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. Each of these recombinants were found to be less pathogenic than the parental virus M41-CK as shown in FIGS. 4-6.

Example 3—Vaccination/Challenge Study with M41-R

Candidate vaccine viruses are tested in studies in which fertilized chicken eggs are vaccinated in ovo at 19 days embryonation and in which the hatchability of the inoculated eggs is determined. The clinical health of the chickens is investigated and the chickens are challenged at 21 days of age with a virulent IB M41 challenge virus at 103.65 EID50 per dose.

Clinical signs are investigated after challenge protection by the vaccine and a ciliostasis test is 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).

TABLE 3 Between hatch and End of Treatment Eggs At hatch challenge Challenge study Saline MDA Determine Clinical Chickens 5 days post IB M41- MDA hatch examination at age of challenge a K-A- percent- of birds 21 days ciliostasis ADRP ages test will be IB M41- MDA done K-S- ADRP IB M41- MDA K-3ab In ovo vaccination of 19 days embryonated eggs was performed by hand with needles of the same size as the automatic equipment. After housing the different treatment groups are housed in separate units. The clinical health of the chickens is examined daily. After the challenge the clinical health of the chickens will be examined daily.

Results of Hatchability Study

SPF broiler eggs were incubated according to routine incubation procedures. At 19 days incubation 6 groups of 30 eggs were inoculated with 104 EID50 per dose of the IB vaccine strains IB M41-K-A-ADRP, IB M41-K-S-ADRP and IB M41-K-3ab. Another group of 30 eggs was inoculated with a placebo, i.e. PBS. At 21 days incubation the eggs hatched and the following results were obtained:

    • PBS 87% (26/30) hatched, 1 chick died after hatching and 3 eggs remained unopened and unmarked for embryo injection.
    • M41-K-A-ARDP 97% (29/30) hatched, 1 egg remained unopened and unmarked for embryo injection.
    • M41-K-S-ARDP 50% (15/30) hatched, 4 eggs remained unopened of which 1 was marked for embryo injection. The other eggs/animals were not viable.
    • M41-K-3ab 47% (18/30) hatched, 7 eggs remained unopened of which 3 were marked for embryo injection. The other eggs/animals were not viable.

IB M41-K-A-ADRP demonstrated good hatchability of inoculated eggs and so is considered suitable as a vaccine for in ovo vaccination. This strain will be tested for efficacy in a challenge test as detailed above.

All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described methods and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in molecular biology, virology or related fields are intended to be within the scope of the following claims.

Claims

1. A live, attenuated coronavirus comprising a mutation in non-structural protein nsp-3 and/or deletion of accessory proteins 3a and/or 3b.

2. The coronavirus according to claim 1 wherein the mutation in nsp-3 is in the adenosine diphosphate-ribose-1′-phosphatase (ADRP) region.

3. The coronavirus according to claim 1, wherein the nsp-3 gene encodes a protein comprising one or more amino acid mutations selected from the list of:

a) Asn (N) to Ala (A) at position 373 in SEQ ID NO: 6; and
b) Gly (G) to Ser (S) at position 379 in SEQ ID NO: 6.

4. The coronavirus according to claim 1, wherein i) the nsp-3 gene encodes a protein comprising the amino acid mutation Asn (N) to Ala (A) at position 373 in SEQ ID NO: 6; or

ii) the nsp-3 gene encodes a protein comprising the amino acid mutation Gly (G) to Ser (S) at position 379 in SEQ ID NO: 6.

5. (canceled)

6. The coronavirus according to claim 1 wherein the nsp-3 gene comprises one or more nucleotide substitutions selected from the list of:

a) A to G at nucleotide position 1116 and A to C at nucleotide position 1117 compared to the sequence shown as SEQ ID NO: 5; and
b) G to A at nucleotide position 1138 compared to the sequence shown as SEQ ID NO: 5.

7. A coronavirus according to claim 1 comprising a deletion from nucleotide position 37 to 384 of the sequence shown as SEQ ID NO: 2.

8. The coronavirus according to claim 1 which is an infectious bronchitis virus (IBV), optionally wherein the IBV is M41.

9. (canceled)

10. The coronavirus according to claim 8, which comprises an S protein wherein at least part of which is from an IBV serotype other than M41.

11. The coronavirus according to claim 10, wherein the S1 subunit is from an IBV serotype other than M41.

12. The coronavirus according to claim 1 which has reduced pathogenicity compared to a wild-type coronavirus, such that when the virus is administered to an embryonated egg, it is capable of replicating without being pathogenic to the embryo.

13. A modified nsp-3 gene as defined in claim 1.

14. A protein encoded by a modified nsp-3 gene according to claim 13.

15. A plasmid comprising a modified nsp-3 gene according to claim 13 or a modified gene 3 with a deletion of accessory protein 3a and/or 3b.

16. A method for making the coronavirus according to claim 1 which comprises the following steps:

(i) transfecting a plasmid comprising a modified nsp-3 gene as defined in claim 1 or a modified gene 3 with a deletion of accessory protein 3a and/or 3b into a host cell;
(ii) infecting the host cell with a recombining virus comprising the genome of a coronavirus strain with a nsp-3 gene and/or gene 3;
(iii) allowing homologous recombination to occur between the gene sequences in the plasmid and the corresponding sequences in the recombining virus genome to produce a modified gene; and
(iv) selecting for recombining virus comprising the modified gene.

17. The method according to claim 16, wherein the recombining virus is a vaccinia virus.

18. The method according to claim 16 which also includes the step:

(v) recovering recombinant coronavirus comprising the modified gene from the DNA from the recombining virus from step (iv).

19. A cell capable of producing a coronavirus according to claim 1.

20. A vaccine comprising a coronavirus according to claim 1 and a pharmaceutically acceptable carrier.

21. A method for treating and/or preventing a disease in a subject which comprises the step of administering a vaccine according to claim 20 to the subject.

22-23. (canceled)

24. The method according to claim 21 wherein the disease is infectious bronchitis (IB).

25. The method according to claim 21 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.

26. The method according to claim 24 wherein the vaccination is in ovo vaccination.

27. A method for producing a vaccine according to claim 20, which comprises the step of infecting a cell according to claim 19 with a coronavirus according to claim 1.

Patent History
Publication number: 20190022214
Type: Application
Filed: Jan 26, 2017
Publication Date: Jan 24, 2019
Inventors: Erica Bickerton (Pirbright, Woking), Sarah Keep (Pirbright, Woking), Paul Britton (Pirbright, Woking)
Application Number: 16/071,938
Classifications
International Classification: A61K 39/215 (20060101); A61P 31/12 (20060101);