Attenuated Infectious Bronchitis Virus

Abstract

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.

Description

FIELD OF THE INVENTION

[0001] 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

[0002] 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.

[0003] 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.

[0004] 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.

[0005] 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.

[0006] 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.

[0007] 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

[0008] 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.

[0009] 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.

[0010] 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.

[0011] The mutation in nsp-3 may be in the adenosine diphosphate-ribose-1'-phosphatase (ADRP) region.

[0012] The modified nsp-3 gene may encode a protein comprising one or more amino acid mutations selected from the list of:

[0013] a) Asn (N) to Ala (A) at position 373 in SEQ ID NO: 6; and

[0014] b) Gly (G) to Ser (S) at position 379 in SEQ ID NO: 6.

[0015] 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.

[0016] 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.

[0017] The modified nsp-3 gene may comprise one or more nucleotide substitutions selected from the list of:

[0018] 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

[0019] b) G to A at nucleotide position 1138 compared to the sequence shown as SEQ ID NO: 5.

[0020] The live attenuated coronavirus may comprise a deletion from nucleotide position 37 to 384 of the sequence shown as SEQ ID NO: 2.

[0021] The coronavirus may be an infectious bronchitis virus (IBV).

[0022] The coronavirus may be IBV M41.

[0023] The coronavirus may comprise an S protein wherein at least part of which is from an IBV serotype other than M41.

[0024] For example, the S1 subunit or the entire S protein may be from an IBV serotype other than M41.

[0025] 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.

[0026] In a second aspect, the present invention provides a modified nsp-3 gene as defined in connection with the first aspect of the invention.

[0027] 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.

[0028] 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.

[0029] 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:

[0030] (i) transfecting a plasmid according to the fourth aspect of the invention into a host cell;

[0031] (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;

[0032] (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

[0033] (iv) selecting for recombining virus comprising the modified gene.

[0034] The recombining virus may be a vaccinia virus.

[0035] The method may also include the step:

[0036] (v) recovering recombinant coronavirus comprising the modified gene from the DNA from the recombining virus from step (iv).

[0037] In a sixth aspect, the present invention provides a cell capable of producing a coronavirus according to the first aspect of the invention.

[0038] 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.

[0039] 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.

[0040] Further aspects of the invention provide:

[0041] the vaccine according to the seventh aspect of the invention for use in treating and/or preventing a disease in a subject.

[0042] 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.

[0043] The disease may be infectious bronchitis (IB).

[0044] 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.

[0045] Vaccination may be by in ovo vaccination.

[0046] 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

[0047] FIG. 1--plasmid used to produce rIBV M41K-S-ADRP.

[0048] FIG. 2--plasmid used to produce rIBV M41K-A-ADRP.

[0049] FIG. 3--plasmid used to produce rIBV M41K-del3ab.

[0050] 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).

[0051] FIG. 5--Summary of clinical signs, virus detection and isolation on day 4 post-infection.

[0052] FIG. 6--Summary of clinical signs, virus detection and isolation on day 6 post-infection.

[0053] 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

[0054] 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

[0055] The present invention provides a mutant coronavirus which has reduced pathogenicity compared to a corresponding wild type coronavirus.

[0056] Coronavirus

[0057] 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.

[0058] The genomic size of coronaviruses ranges from approximately 27 to 32 kilobases, which is the longest size for any known RNA virus.

[0059] 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.

[0060] 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.

[0061] 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.

[0062] Mouse hepatitis virus (MHV) is a coronavirus that causes an epidemic murine illness with high mortality, especially among colonies of laboratory mice.

[0063] Coronaviruses are divided into four groups, as shown below:

[0064] Alpha

[0065] Canine coronavirus (CCoV)

[0066] Feline coronavirus (FeCoV)

[0067] Human coronavirus 229E (HCoV-229E)

[0068] Porcine epidemic diarrhoea virus (PEDV)

[0069] Transmissible gastroenteritis virus (TGEV)

[0070] Human Coronavirus NL63 (NL or New Haven)

[0071] Beta

[0072] Bovine coronavirus (BCoV)

[0073] Canine respiratory coronavirus (CRCoV)--Common in SE Asia and Micronesia

[0074] Human coronavirus OC43 (HCoV-OC43)

[0075] Mouse hepatitis virus (MHV)

[0076] Porcine haemagglutinating encephalomyelitis virus (HEV)

[0077] 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.

[0078] (No common name as of yet) (HCoV-HKU1)

[0079] Severe acute respiratory syndrome coronavirus (SARS-CoV)

[0080] Middle East respiratory syndrome coronavirus (MERS-CoV)

[0081] Gamma

[0082] Infectious bronchitis virus (IBV)

[0083] Turkey coronavirus (Bluecomb disease virus)

[0084] Pheasant coronavirus

[0085] Guinea fowl coronavirus

[0086] Delta

[0087] Bulbul coronavirus (BuCoV)

[0088] Thrush coronavirus (ThCoV)

[0089] Munia coronavirus (MuCoV)

[0090] Porcine coronavirus (PorCov) HKU15

[0091] 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.

[0092] 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.

[0093] IBV

[0094] 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.

[0095] 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.

[0096] 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.

[0097] 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.

[0098] 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.

[0099] 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.

[0100] 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.

[0101] 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.

[0102] 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 .about.500 nucleotides in IBV).

[0103] 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.

[0104] 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.

[0105] 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.TM.). 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.

[0106] The genome sequence of IBV M41-CK is provided as SEQ ID NO: 1.

TABLE-US-00001 IBV M41-CK Sequence SEQ ID NO: 1 ACTTAAGATAGATATTAATATATATCTATCACACTAGCCTTGCGCTAGATTTCCAACTTAACAAAACGGACTTA- AATACCTACAGCTGGTCCT CATAGGTGTTCCATTGCAGTGCACTTTAGTGCCCTGGATGGCACCTGGCCACCTGTCAGGTTTTTGTTATTAAA- ATCTTATTGTTGCTGGTAT CACTGCTTGTTTTGCCGTGTCTCACTTTATACATCCGTTGCTTGGGCTACCTAGTATCCAGCGTCCTACGGGCG- CCGTGGCTGGTTCGAGTGC GAAGAACCTCTGGTTCATCTAGCGGTAGGCGGGTGTGTGGAAGTAGCACTTCAGACGTACCGGTTCTGTTGTGT- GAAATACGGGGTCACCTCC CCCCACATACCTCTAAGGGCTTTTGAGCCTAGCGTTGGGCTACGTTCTCGCATAAGGTCGGCTATACGACGTTT- GTAGGGGGTAGTGCCAAAC AACCCCTGAGGTGACAGGTTCTGGTGGTGTTTAGTGAGCAGACATACAATAGACAGTGACAACATGGCTTCAAG- CCTAAAACAGGGAGTATCT CCCAAACTAAGGGATGTCATTCTTGTATCCAAAGACATTCCTGAACAACTTTGTGACGCTTTGTTTTTCTATAC- GTCACACAACCCTAAGGAT TACGCTGATGCTTTTGCAGTTAGGCAGAAGTTTGATCGTAATCTGCAGACTGGGAAACAGTTCAAATTTGAAAC- TGTGTGTGGTCTCTTCCTC TTGAAGGGAGTTGACAAAATAACACCTGGCGTCCCAGCAAAAGTCTTAAAAGCCACTTCTAAGTTGGCAGATTT- AGAAGACATCTTTGGTGTC TCTCCCTTTGCAAGAAAATATCGTGAACTTTTGAAGACAGCATGCCAGTGGTCTCTTACTGTAGAAACACTGGA- TGCTCGTGCACAAACTCTT GATGAAATTTTTGACCCTACTGAAATACTTTGGCTTCAGGTGGCAGCAAAAATCCAAGTTTCGGCTATGGCGAT- GCGCAGGCTTGTTGGAGAA GTAACTGCAAAAGTCATGGATGCTTTGGGCTCAAATATGAGTGCTCTTTTCCAGATTTTTAAACAACAAATAGT- CAGAATTTTTCAAAAAGCG CTGGCTATTTTTGAGAATGTGAGTGAATTACCACAGCGTATTGCAGCACTTAAGATGGCTTTTGCTAAGTGTGC- CAAGTCCATTACTGTTGTG GTTATGGAGAGGACTCTAGTTGTTAGAGAGTTCGCAGGAACTTGTCTTGCAAGCATTAATGGTGCTGTTGCAAA- ATTCTTTGAAGAACTCCCA AATGGTTTCATGGGTGCTAAAATTTTCACTACACTTGCCTTCTTTAGGGAGGCTGCAGTGAAAATTGTGGATAA- CATACCAAATGCACCGAGA GGCACTAAAGGGTTTGAAGTCGTTGGTAATGCCAAAGGTACACAAGTTGTTGTGCGTGGCATGCGAAATGACTT- AACACTGCTTGACCAAAAA GCTGAAATTCCTGTGGAGTCAGAAGGTTGGTCTGCAATTTTGGGTGGACATCTTTGCTATGTCTTTAAGAGTGG- TGATCGCTTTTACGCGGCA CCTCTTTCAGGAAATTTTGCATTGCATGATGTGCATTGTTGTGAGCGTGTTGTCTGTCTTTCTGATGGTGTAAC- ACCGGAGATAAATGATGGA CTTATTCTTGCAGCAATCTACTCTTCTTTTAGTGTCGCAGAACTTGTGGCAGCCATTAAAAGGGGTGAACCATT- TAAGTTTCTGGGTCATAAA TTTGTGTATGCAAAGGATGCAGCAGTTTCTTTTACATTAGCGAAGGCTGCTACTATTGCAGATGTTTTGAAGCT- GTTTCAATCAGCGCGTGTG AAAGTAGAAGATGTTTGGTCTTCACTTACTGAAAAGTCTTTTGAATTCTGGAGGCTTGCATATGGAAAAGTGCG- TAATCTCGAAGAATTTGTT AAGACTTGTTTTTGTAAGGCTCAAATGGCGATTGTGATTTTAGCGACAGTGCTTGGAGAGGGCATTTGGCATCT- TGTTTCGCAAGTCATCTAT AAAGTAGGTGGTCTTTTTACTAAAGTTGTTGACTTTTGTGAAAAATATTGGAAAGGTTTTTGTGCACAGTTGAA- AAGAGCTAAGCTCATTGTC ACTGAAACCCTCTGTGTTTTGAAAGGAGTTGCACAGCATTGTTTTCAACTATTGCTGGATGCAATACAGTTTAT- GTATAAAAGTTTTAAGAAG TGTGCACTTGGTAGAATCCATGGAGACTTGCTCTTCTGGAAAGGAGGTGTGCACAAAATTATTCAAGAGGGCGA- TGAAATTTGGTTTGACGCC ATTGATAGTATTGATGTTGAAGATCTGGGTGTTGTTCAAGAAAAATTGATTGATTTTGATGTTTGTGATAATGT- GACACTTCCAGAGAACCAA CCCGGTCATATGGTTCAAATCGAGGATGACGGAAAGAACTACATGTTCTTCCGCTTCAAAAAGGATGAGAACAT- TTATTATACACCAATGTCA CAGCTTGGTGCTATTAATGTGGTTTGCAAAGCAGGCGGTAAAACTGTCACCTTTGGAGAAACTACTGTGCAAGA- AATACCACCACCTGATGTT GTGTTTATTAAGGTTAGCATTGAGTGTTGTGGTGAACCATGGAATACAATCTTCAAAAAGGCTTATAAGGAGCC- CATTGAAGTAGAGACAGAC CTCACAGTTGAACAATTGCTCTCTGTGGTCTATGAGAAAATGTGTGATGATCTCAAGCTGTTTCCGGAGGCTCC- AGAACCACCACCATTTGAG AATGTCACACTTGTTGATAAGAATGGTAAAGATTTGGATTGCATAAAATCATGCCATCTGATCTATCGTGATTA- TGAGAGCGATGATGACATC GAGGAAGAAGATGCAGAAGAATGTGACACGGATTCAGGTGATGCTGAGGAGTGTGACACTAATTCAGAATGTGA- AGAAGAAGATGAGGATACT AAAGTGTTGGCTCTTATACAAGACCCGGCAAGTAACAAATATCCTCTGCCTCTTGATGATGATTATAGCGTCTA- CAATGGATGTATTGTTCAT AAGGACGCTCTCGATGTTGTGAATTTACCATCTGGTGAAGAAACCTTTGTTGTCAATAACTGCTTTGAAGGGGC- TGTTAAAGCTCTTCCGCAG AAAGTTATTGATGTTCTAGGTGACTGGGGTGAGGCTGTTGATGCGCAAGAACAATTGTGTCAACAAGAATCAAC- TCGGGTCATATCTGAGAAA TCAGTTGAGGGTTTTACTGGTAGTTGTGATGCAATGGCTGAACAAGCTATTGTTGAAGAGCAGGAAATAGTACC- TGTTGTTGAACAAAGTCAG GATGTAGTTGTTTTTACACCTGCAGACCTAGAAGTTGTTAAAGAAACAGCAGAAGAGGTTGATGAGTTTATTCT- CATTTCTGCTGTCCCTAAA GAAGAAGTTGTGTCTCAGGAGAAAGAGGAGCCACAGGTTGAGCAAGAGCCTACCCTAGTTGTTAAAGCACAACG- TGAGAAGAAGGCTAAAAAG TTCAAAGTTAAACCAGCTACATGTGAAAAACCCAAATTTTTGGAGTACAAAACATGTGTGGGTGATTTGGCTGT- TGTAATTGCCAAAGCATTG GATGAGTTTAAAGAGTTCTGCATTGTAAACGCTGCAAATGAGCACATGTCGCATGGTGGTGGCGTTGCAAAGGC- AATTGCAGACTTTTGTGGA CCGGACTTTGTTGAATATTGCGCGGACTATGTTAAGAAACATGGTCCACAGCAAAAACTTGTCACACCTTCATT- TGTTAAAGGCATTCAATGT GTGAATAATGTTGTAGGACCTCGCCATGGAGACAGCAACTTGCGTGAGAAGCTTGTTGCTGCTTACAAGAGTGT- TCTTGTAGGTGGAGTGGTT AACTATGTTGTGCCAGTTCTCTCATCAGGGATTTTTGGTGTAGATTTTAAAATATCAATAGATGCTATGCGCGA- AGCTTTTAAAGGTTGTGCC ATACGCGTTCTTTTATTTTCTCTGAGTCAAGAACACATCGATTATTTCGATGCAACTTGTAAGCAGAAGACAAT- TTATCTTACGGAGGATGGT GTTAAATACCGCTCTGTTGTTTTAAAACCTGGTGATTCTTTGGGTCAATTTGGACAGGTTTTTGCAAGAAATAA- GGTAGTCTTTTCGGCTGAT GATGTTGAGGATAAAGAAATCCTCTTTATACCCACAACTGACAAGACTATTCTTGAATATTATGGTTTAGATGC- GCAAAAGTATGTAACATAT TTGCAAACGCTTGCGCAGAAATGGGATGTTCAATATAGAGACAATTTTGTTATATTAGAGTGGCGTGACGGAAA- TTGCTGGATTAGTTCAGCA ATAGTTCTCCTTCAAGCTGCTAAAATTAGATTTAAAGGTTTTCTTGCAGAAGCATGGGCTAAACTGTTGGGTGG- AGATCCTACAGACTTTGTT GCCTGGTGTTATGCAAGTTGCAATGCTAAAGTAGGTGATTTTTCAGATGCTAATTGGCTTTTGGCCAATTTAGC- AGAACATTTTGACGCAGAT TACACAAATGCACTTCTTAAGAAGTGTGTGTCGTGCAATTGTGGTGTTAAGAGTTATGAACTTAGGGGTCTTGA- AGCCTGTATTCAGCCAGTT CGAGCACCTAATCTTCTACATTTTAAAACGCAATATTCAAATTGCCCAACCTGTGGTGCAAGTAGTACGGATGA- AGTAATAGAAGCTTCATTA CCGTACTTATTGCTTTTTGCTACTGATGGTCCTGCTACAGTTGATTGTGATGAAAATGCTGTAGGGACTGTTGT- TTTCATTGGCTCTACTAAT AGTGGCCATTGTTATACACAAGCCGATGGTAAGGCTTTTGACAATCTTGCTAAGGATAGAAAATTTGGAAGGAA- GTCGCCTTACATTACAGCA ATGTATACACGTTTTTCTCTTAGGAGTGAAAATCCCCTACTTGTTGTTGAACATAGTAAGGGTAAAGCTAAAGT- AGTAAAAGAAGATGTTTCT AACCTTGCTACTAGTTCTAAAGCCAGTTTTGACGATCTTACTGACTTTGAACAGTGGTATGATAGCAACATCTA- TGAGAGTCTTAAAGTGCAG GAGACACCTGATAATCTTGATGAATATGTGTCATTTACGACAAAGGAAGATTCTAAGTTGCCACTGACACTTAA- AGTTAGAGGTATCAAATCA GTTGTTGACTTTAGGTCTAAGGATGGTTTTACTTATAAGTTAACACCTGATACTGATGAAAATTCAAAAACACC- AGTCTACTACCCAGTCTTG GATTCTATTAGTCTTAGGGCAATATGGGTTGAAGGCAGTGCTAATTTTGTTGTTGGGCATCCAAATTATTATAG- TAAGTCTCTCCGAATTCCC ACGTTTTGGGAAAATGCCGAGAGCTTTGTTAAAATGGGTTATAAAATTGATGGTGTAACTATGGGCCTTTGGCG- TGCAGAACACCTTAATAAA CCTAATTTGGAGAGAATTTTTAACATTGCTAAGAAAGCTATTGTTGGATCTAGTGTTGTTACTACGCAGTGTGG- TAAAATACTAGTTAAAGCA GCTACATACGTTGCCGATAAAGTAGGTGATGGTGTAGTTCGCAATATTACAGATAGAATTAAGGGTCTTTGTGG- ATTCACACGTGGCCATTTT GAAAAGAAAATGTCCCTACAATTTCTAAAGACACTTGTGTTCTTTTTCTTTTATTTCTTAAAGGCTAGTGCTAA- GAGTTTAGTTTCTAGCTAT AAGATTGTGTTATGTAAGGTGGTGTTTGCTACCTTACTTATAGTGTGGTTTATATACACAAGTAATCCAGTAGT- GTTTACTGGAATACGTGTG CTAGACTTCCTATTTGAAGGTTCTTTATGTGGTCCTTATAATGACTACGGTAAAGATTCTTTTGATGTGTTACG- CTATTGTGCAGGTGATTTT ACTTGTCGTGTGTGTTTACATGATAGAGATTCACTTCATCTGTACAAACATGCTTATAGCGTAGAACAAATTTA- TAAGGATGCAGCTTCTGGC ATTAACTTTAATTGGAATTGGCTTTATTTGGTCTTTCTAATATTATTTGTTAAGCCAGTGGCAGGTTTTGTTAT- TATTTGTTATTGTGTTAAG TATTTGGTATTGAGTTCAACTGTGTTGCAAACTGGTGTAGGTTTTCTAGATTGGTTTGTAAAAACAGTTTTTAC- CCATTTTAATTTTATGGGA GCGGGATTTTATTTCTGGCTCTTTTACAAGATATACGTACAAGTGCATCATATATTGTACTGTAAGGATGTAAC- ATGTGAAGTGTGCAAGAGA GTTGCACGCAGCAACAGGCAAGAGGTTAGCGTTGTAGTTGGTGGACGCAAGCAAATAGTGCATGTTTACACTAA- TTCTGGCTATAACTTTTGT AAGAGACATAATTGGTATTGTAGAAATTGTGATGATTATGGTCACCAAAATACATTTATGTCCCCTGAAGTTGC- TGGCGAGCTTTCTGAAAAG CTTAAGCGCCATGTTAAACCTACAGCATATGCTTACCACGTTGTGTATGAGGCATGCGTGGTTGATGATTTTGT- TAATTTAAAATATAAGGCT GCAATTCCTGGTAAGGATAATGCATCTTCTGCTGTTAAGTGTTTCAGTGTTACAGATTTTTTAAAGAAAGCTGT- TTTTCTTAAGGAGGCATTG AAATGTGAACAAATATCTAATGATGGTTTTATAGTGTGTAATACACAGAGTGCGCATGCACTAGAGGAAGCAAA- GAATGCAGCCGTCTATTAT GCGCAATATCTGTGTAAGCCAATACTTATACTTGACCAGGCACTTTATGAGCAATTAATAGTAGAGCCTGTGTC- TAAGAGTGTTATAGATAAA GTGTGTAGCATTTTGTCTAATATAATATCTGTAGATACTGCAGCTTTAAATTATAAGGCAGGCACACTTCGTGA- TGCTCTGCTTTCTATTACT AAAGACGAAGAAGCCGTAGATATGGCTATCTTCTGCCACAATCATGAAGTGGAATACACTGGTGACGGTTTTAC- TAATGTGATACCGTCATAT GGTATGGACACTGATAAGTTGACACCTCGTGATAGAGGGTTTTTGATAAATGCAGATGCTTCTATTGCTAATTT- AAGAGTCAAAAATGCTCCT CCGGTAGTATGGAAGTTTTCTGATCTTATTAAATTGTCTGACAGTTGCCTTAAATATTTAATTTCAGCTACTGT- CAAGTCAGGAGGTCGTTTC TTTATAACAAAGTCTGGTGCTAAACAAGTTATTTCTTGTCATACCCAGAAACTGTTGGTAGAGAAAAAGGCAGG- TGGTGTTATTAATAACACT TTTAAATGGTTTATGAGTTGTTTTAAATGGCTTTTTGTCTTTTATATACTTTTTACAGCATGTTGTTTGGGTTA- CTACTATATGGAGATGAAT AAAAGTTTTGTTCACCCCATGTATGATGTAAACTCCACACTGCATGTTGAAGGGTTCAAAGTTATAGACAAAGG- TGTTATTAGAGAGATTGTG TCAGAAGATAATTGTTTCTCTAATAAGTTTGTTAATTTTGACGCCTTTTGGGGTAAATCATATGAAAATAATAA- AAACTGTCCAATTGTTACA GTTGTTATAGATGGTGACGGGACAGTAGCTGTTGGTGTTCCTGGTTTTGTATCATGGGTTATGGATGGTGTTAT- GTTTGTGCATATGACACAG

ACTGATCGTAGACCTTGGTACATTCCTACCTGGTTTAATAGAGAAATTGTTGGTTACACTCAGGATTCAATTAT- CACTGAGGGTAGTTTTTAT ACATCTATAGCATTATTTTCTGCTAGATGTTTATATTTAACAGCCAGCAATACACCTCAATTGTATTGTTTTAA- TGGCGACAATGATGCACCT GGAGCCTTACCATTTGGTAGTATTATTCCTCATAGAGTATACTTCCAACCTAATGGTGTTAGGCTTATAGTTCC- ACAACAAATACTGCATACA CCCTACATAGTGAAGTTTGTTTCAGACAGCTATTGTAGAGGTAGTGTATGTGAGTATACTAAACCAGGTTACTG- TGTGTCACTAGACTCCCAA TGGGTTTTGTTTAATGATGAATACATTAGTAAACCTGGCGTTTTCTGTGGTTCTACTGTTAGAGAACTTATGTT- TAATATGGTTAGTACATTC TTTACTGGTGTCAACCCTAATATTTATATTCAGCTAGCAACTATGTTTTTAATACTAGTTGTTATTGTGTTAAT- TTTTGCAATGGTTATAAAG TTTCAAGGTGTTTTTAAAGCTTATGCGACCATTGTGTTTACAATAATGTTAGTTTGGGTTATTAATGCATTTGT- TTTGTGTGTACATAGTTAT AATAGTGTTTTAGCTGTTATATTATTAGTACTCTATTGCTATGCATCATTGGTTACAAGTCGCAATACTGCTAT- AATAATGCATTGTTGGCTT GTTTTTACCTTTGGTTTAATAGTACCCACATGGTTGGCTTGTTGCTATCTGGGATTTATTCTTTATATGTACAC- ACCGTTGGTTTTCTGGTGT TACGGTACTACTAAAAATACTCGTAAGTTGTATGATGGCAACGAGTTTGTTGGTAATTATGACCTTGCTGCGAA- GAGCACTTTTGTTATTCGT GGTACTGAATTTGTTAAGCTTACGAATGAGATAGGTGATAAATTTGAAGCCTATCTTTCTGCGTATGCTAGACT- TAAATACTATTCAGGCACT GGTAGTGAGCAAGATTACTTGCAAGCTTGTCGTGCATGGTTAGCTTATGCTTTGGACCAATATAGAAATAGTGG- TGTTGAGGTTGTTTATACC CCACCGCGTTACTCTATTGGTGTTAGTAGACTACACGCTGGTTTTAAAAAACTAGTTTCTCCTAGTAGTGCTGT- TGAGAAGTGCATTGTTAGT GTCTCTTATAGAGGCAATAATCTTAATGGACTGTGGCTGGGTGATTCTATTTACTGCCCACGCCATGTGTTAGG- TAAGTTTAGTGGTGACCAG TGGGGTGACGTACTAAACCTTGCTAATAATCATGAGTTTGAAGTTGTAACTCAAAATGGTGTTACTTTGAATGT- TGTCAGCAGGCGGCTTAAA GGAGCAGTTTTAATTTTACAAACTGCAGTTGCCAATGCTGAAACTCCTAAGTATAAGTTTGTTAAAGCTAATTG- TGGTGATAGTTTCACTATA GCTTGTTCTTATGGTGGTACAGTTATAGGACTTTACCCTGTCACTATGCGTTCTAATGGTACTATTAGAGCATC- TTTCCTAGCAGGAGCCTGT GGCTCAGTTGGTTTTAATATAGAAAAGGGTGTAGTTAATTTCTTTTATATGCACCATCTTGAGTTACCTAATGC- ATTACACACTGGAACTGAC CTAATGGGTGAGTTTTATGGTGGTTATGTAGATGAAGAGGTTGCGCAAAGAGTGCCACCAGATAATCTAGTTAC- TAACAATATTGTAGCATGG CTCTATGCGGCAATTATTAGTGTTAAAGAAAGTAGTTTTTCACAACCTAAATGGTTGGAGAGTACTACTGTTTC- TATTGAAGATTACAATAGG TGGGCTAGTGATAATGGTTTTACTCCATTTTCCACTAGTACTGCTATTACTAAATTAAGTGCTATAACTGGGGT- TGATGTTTGTAAACTCCTT CGCACTATTATGGTAAAAAGTGCTCAATGGGGTAGTGATCCCATTTTAGGACAATATAATTTTGAAGACGAATT- GACACCAGAATCTGTATTT AATCAAGTTGGTGGTGTTAGGTTACAGTCTTCTTTTGTAAGAAAAGCTACATCTTGGTTTTGGAGTAGATGTGT- ATTAGCTTGCTTCTTGTTT GTGTTGTGTGCTATTGTCTTATTTACGGCAGTGCCACTTAAGTTTTATGTACATGCAGCTGTTATTTTGTTGAT- GGCTGTGCTCTTTATTTCT TTTACTGTTAAACATGTTATGGCATACATGGACACTTTCCTATTGCCTACATTGATTACAGTTATTATTGGAGT- TTGTGCTGAAGTCCCTTTC ATATACAATACTCTAATTAGTCAAGTTGTTATTTTCTTAAGCCAATGGTATGATCCTGTAGTCTTTGATACTAT- GGTACCATGGATGTTATTG CCATTAGTGTTGTACACTGCTTTTAAGTGTGTACAAGGCTGCTATATGAATTCTTTCAATACTTCTTTGTTAAT- GCTGTATCAGTTTATGAAG TTAGGTTTTGTTATTTACACCTCTTCAAACACTCTTACTGCATATACAGAAGGTAATTGGGAGTTATTCTTTGA- GTTGGTTCACACTATTGTG TTGGCTAATGTTAGTAGTAATTCCTTAATTGGTTTAATTGTTTTTAAGTGTGCTAAGTGGATTTTATATTATTG- CAATGCAACATACTTTAAT AATTATGTGTTAATGGCAGTCATGGTTAATGGCATAGGCTGGCTTTGCACCTGTTACTTTGGATTGTATTGGTG- GGTTAATAAAGTTTTTGGT TTAACCTTAGGTAAATACAATTTTAAAGTTTCAGTAGATCAATATAGGTATATGTGTTTGCATAAGGTAAATCC- ACCTAAAACTGTGTGGGAG GTCTTTACTACAAATATACTTATACAAGGAATTGGAGGCGATCGTGTGTTGCCTATAGCTACAGTGCAATCTAA- ATTGAGTGATGTAAAGTGT ACAACTGTTGTTTTAATGCAGCTTTTGACTAAGCTTAATGTTGAAGCAAATTCAAAAATGCATGCTTATCTTGT- TGAGTTACACAATAAAATC CTCGCATCTGATGATGTTGGAGAGTGCATGGATAATTTATTGGGTATGCTTATAACACTATTTTGTATAGATTC- TACTATTGATTTGGGTGAG TATTGTGATGATATACTTAAGAGGTCAACTGTATTACAATCGGTTACTCAAGAGTTTTCGCACATACCCTCGTA- TGCTGAATATGAAAGAGCT AAGAGTATTTATGAAAAGGTTTTAGCCGATTCTAAAAATGGTGGTGTAACACAGCAAGAGCTTGCTGCATATCG- TAAAGCTGCCAATATTGCA AAGTCAGTTTTTGATAGAGACTTGGCTGTTCAAAAGAAGTTAGATAGCATGGCAGAACGTGCTATGACAACAAT- GTATAAAGAGGCGCGTGTA ACTGATAGAAGAGCAAAATTAGTTTCATCATTACATGCACTACTTTTTTCAATGCTTAAGAAAATAGATTCTGA- GAAGCTTAATGTCTTATTT GACCAGGCGAATAGTGGTGTTGTACCCCTAGCAACTGTTCCAATTGTTTGTAGTAATAAGCTTACCCTTGTTAT- ACCAGACCCAGAGACGTGG GTCAAGTGTGTGGAGGGTGTGCATGTTACATATTCAACAGTTGTTTGGAATATAGACTGTGTTACTGATGCCGA- TGGCACAGAGTTACACCCC ACTTCTACAGGTAGTGGATTGACTTACTGTATAAGTGGTGATAATATAGCATGGCCTTTAAAGGTTAACTTGAC- TAGGAATGGGCATAATAAG GTTGATGTTGCCTTGCAAAATAATGAGCTTATGCCTCACGGTGTAAAGACAAAGGCTTGCGTAGCAGGTGTAGA- TCAAGCACATTGTAGCGTT GAGTCTAAATGTTATTATACAAGTATTAGTGGCAGTTCAGTTGTAGCTGCTATTACCTCTTCAAATCCTAATCT- GAAAGTAGCCTCTTTTTTG AATGAGGCAGGTAATCAGATTTATGTAGACTTAGACCCACCATGTAAATTTGGTATGAAAGTGGGTGATAAGGT- TGAAGTTGTTTACCTGTAT TTTATAAAAAATACGAGGTCTATTGTAAGAGGTATGGTACTTGGTGCTATATCTAATGTTGTTGTGTTACAATC- TAAAGGTCATGAGACAGAG GAAGTGGATGCTGTAGGCATTCTCTCACTTTGTTCTTTTGCAGTAGATCCTGCGGATACATATTGTAAATATGT- GGCAGCAGGTAATCAACCT TTAGGTAACTGTGTTAAAATGTTGACAGTACATAATGGTAGTGGTTTTGCAATAACATCAAAGCCAAGTCCAAC- TCCGGATCAGGATTCTTAT GGAGGAGCTTCTGTGTGTCTTTATTGTAGAGCACATATAGCACACCCTGGCGGAGCAGGAAATTTAGATGGACG- CTGTCAATTTAAAGGTTCT TTTGTGCAAATACCTACTACGGAGAAAGATCCTGTTGGATTCTGTCTACGTAACAAGGTTTGCACTGTTTGTCA- GTGTTGGATTGGTTATGGA TGTCAGTGTGATTCACTTAGACAACCTAAACCTTCTGTTCAGTCAGTTGCTGTTGCATCTGGTTTTGATAAGAA- TTATTTAAACGGGTACGGG GTAGCAGTGAGGCTCGGCTGATACCCCTAGCTAATGGATGTGACCCCGATGTTGTAAAGCGAGCCTTTGATGTT- TGTAATAAGGAATCAGCCG GTATGTTTCAAAATTTGAAGCGTAACTGTGCACGATTCCAAGAAGTACGTGATACTGAAGATGGAAATCTTGAG- TATTGTGATTCTTATTTTG TGGTTAAACAAACCACTCCTAGTAATTATGAACATGAGAAAGCTTGTTATGAAGACTTAAAGTCAGAAGTAACA- GCTGATCATGATTTCTTTG TGTTCAATAAGAACATTTATAATATTAGTAGGCAGAGGCTTACTAAGTATACTATGATGGATTTTTGCTATGCT- TTGCGGCACTTTGACCCAA AGGATTGCGAAGTTCTTAAAGAAATACTTGTCACTTATGGTTGTATAGAAGATTATCACCCTAAGTGGTTTGAA- GAGAATAAGGATTGGTACG ACCCAATAGAAAACCCTAAATATTATGCCATGTTGGCTAAAATGGGACCTATTGTACGACGTGCTTTATTGAAT- GCTATTGAGTTCGGAAACC TCATGGTTGAAAAAGGTTATGTTGGTGTTATTACACTTGATAACCAAGATCTTAATGGCAAATTTTATGATTTT- GGTGATTTTCAGAAGACAG CGCCTGGTGCTGGTGTTCCTGTTTTTGATACGTATTATTCTTACATGATGCCCATCATAGCCATGACTGATGCG- TTGGCACCTGAGAGGTATT TTGAATATGATGTGCATAAGGGTTATAAATCTTATGATCTCCTCAAGTATGATTATACTGAGGAGAAACAAGAT- TTGTTTCAGAAGTACTTTA AGTATTGGGATCAAGAGTATCACCCTAACTGTCGCGACTGTAGTGATGACAGGTGTTTGATACATTGTGCAAAC- TTCAACATCTTGTTTTCTA CACTTGTACCGCAGACTTCTTTCGGTAATTTGTGTAGAAAGGTTTTTGTTGATGGTGTACCATTTATAGCTACT- TGTGGCTATCATTCTAAGG AACTTGGTGTTATTATGAATCAAGATAACACCATGTCATTTTCAAAAATGGGTTTGAGTCAACTCATGCAGTTT- GTTGGAGATCCTGCCTTGT TAGTGGGGACATCCAATAAATTAGTGGATCTTAGAACGTCTTGTTTTAGTGTTTGTGCTTTAGCGTCTGGTATT- ACTCATCAAACGGTAAAAC CAGGTCACTTTAACAAGGATTTCTACGATTTTGCAGAGAAGGCTGGTATGTTTAAGGAAGGTTCTTCTATACCA- CTTAAACATTTCTTCTACC CACAGACTGGTAATGCTGCTATAAACGATTATGATTATTATCGTTATAACAGGCCTACCATGTTTGATATACGT- CAACTTTTATTTTGTTTAG AAGTGACTTCTAAATATTTTGAATGTTATGAAGGCGGCTGTATACCAGCAAGCCAAGTTGTAGTTAACAATTTA- GATAAGAGTGCAGGTTATC CGTTCAATAAGTTTGGAAAGGCCCGTCTCTATTATGAAATGAGTCTAGAGGAGCAGGACCAACTCTTTGAGAGT- ACAAAGAAGAACGTCCTGC CTACTATAACTCAGATGAATTTAAAATATGCCATATCCGCGAAAAATAGAGCGCGTACAGTGGCAGGTGTGTCT- ATCCTTTCTACTATGACTA ATAGGCAGTTTCATCAGAAGATTCTTAAGTCTATAGTCAACACTAGAAACGCTCCTGTAGTTATTGGAACAACC- AAGTTTTATGGCGGTTGGG ATAACATGTTGAGAAACCTTATTCAGGGTGTTGAAGACCCGATTCTTATGGGTTGGGATTATCCAAAGTGTGAT- AGAGCAATGCCTAATTTGT TGCGTATAGCAGCATCTTTAGTACTCGCTCGTAAACACACTAATTGTTGTACTTGGTCTGAACGCGTTTATAGG- TTGTATAATGAATGCGCTC AGGTTTTATCTGAAACTGTCTTAGCTACAGGTGGTATATATGTGAAACCTGGTGGTACTAGCAGTGGAGATGCT- ACTACTGCTTATGCAAACA GTGTTTTCAACATAATACAAGCCACATCTGCTAATGTTGCGCGTCTTTTGAGTGTTATAACGCGTGATATTGTA- TATGATGACATTAAGAGCT TGCAGTATGAATTGTACCAGCAGGTTTATAGGCGAGTCAATTTTGACCCAGCATTTGTTGAAAAGTTTTATTCT- TATTTGTGTAAGAATTTCT CATTGATGATCTTGTCTGACGACGGTGTTGTTTGTTATAACAACACATTAGCCAAACAAGGTCTTGTAGCAGAT- ATTTCTGGTTTTAGAGAAG TTCTCTACTATCAGAACAATGTTTTTATGGCTGATTCTAAATGTTGGGTTGAACCAGATTTAGAAAAAGGCCCA- CATGAATTTTGTTCACAGC ACACAATGTTAGTGGAGGTTGATGGTGAGCCTAGATACTTGCCATATCCAGACCCATCACGTATTTTGTGTGCA- TGTGTTTTTGTAGATGATT TGGATAAGACAGAATCTGTGGCTGTTATGGAGCGTTATATCGCTCTTGCCATAGATGCGTACCCACTAGTACAT- CATGAAAATGAGGAGTACA AGAAGGTATTCTTTGTGCTTCTTTCATACATCAGAAAACTCTATCAAGAGCTTTCTCAGAATATGCTTATGGAC- TACTCTTTTGTAATGGATA TAGATAAGGGTAGTAAATTTTGGGAACAGGAGTTCTATGAAAATATGTATAGAGCCCCTACAACATTACAGTCT- TGTGGCGTTTGTGTAGTGT GTAATAGTCAAACTATATTGCGCTGTGGTAATTGTATTCGCAAACCATTTTTGTGTTGTAAGTGTTGCTATGAC- CATGTCATGCACACAGACC ACAAAAATGTTTTGTCTATAAATCCTTACATTTGCTCACAGCCAGGTTGTGGTGAAGCAGATGTTACTAAATTG- TACCTCGGAGGTATGTCAT ACTTCTGCGGTAATCATAAACCAAAGTTATCAATACCGTTAGTATCTAATGGTACAGTGTTTGGAATTTACAGG- GCTAATTGTGCAGGTAGCG AAAATGTTGATGATTTTAATCAACTAGCTACTACTAATTGGTCTACTGTGGAACCTTATATTTTGGCAAATCGT-

TGTGTAGATTCGTTGAGAC GCTTTGCTGCAGAGACAGTAAAAGCTACAGAAGAATTACATAAGCAACAATTTGCTAGTGCAGAAGTGAGAGAA- GTACTCTCAGATCGTGAAT TGATTCTGTCTTGGGAGCCAGGTAAAACCAGGCCTCCATTGAATAGAAATTATGTTTTCACTGGCTTTCACTTT- ACTAGAACTAGTAAAGTTC AGCTCGGTGATTTTACATTTGAAAAAGGTGAAGGTAAGGACGTTGTCTATTATCGAGCGACGTCTACTGCTAAA- TTGTCTGTTGGAGACATTT TTGTTTTAACCTCACACAATGTTGTTTCTCTTATAGCGCCAACGTTGTGTCCTCAGCAAACCTTTTCTAGGTTT- GTGAATTTAAGACCTAATG TGATGGTACCTGCGTGTTTTGTAAATAACATTCCATTGTACCATTTAGTAGGCAAGCAGAAGCGTACTACAGTA- CAAGGCCCTCCTGGCAGTG GTAAATCCCATTTTGCTATAGGATTGGCGGCTTACTTTAGTAACGCCCGTGTCGTTTTTACTGCATGCTCTCAT- GCAGCTGTTGATGCTTTAT GTGAAAAAGCTTTTAAGTTTCTTAAAGTAGATGATTGCACTCGTATAGTACCTCAAAGGACTACTATCGATTGC- TTCTCTAAGTTTAAAGCTA ATGACACAGGCAAAAAGTACATTTTTAGTACTATTAATGCCTTGCCAGAAGTTAGTTGTGACATTCTTTTGGTT- GACGAGGTTAGTATGTTGA CCAATTACGAATTGTCTTTTATTAATGGTAAGATAAACTATCAATATGTTGTGTATGTAGGTGATCCTGCTCAA- TTACCGGCGCCTCGTACGT TGCTTAACGGTTCACTCTCTCCAAAGGATTATAATGTTGTCACAAACCTTATGGTTTGTGTTAAACCTGACATT- TTCCTTGCAAAGTGTTACC GTTGTCCTAAAGAAATTGTAGATACTGTTTCTACTCTTGTATATGATGGAAAGTTTATTGCAAATAACCCGGAA- TCACGTCAGTGTTTCAAGG TTATAGTTAATAATGGTAATTCTGATGTAGGACATGAAAGTGGCTCAGCCTACAACATAACTCAATTAGAATTT- GTGAAAGATTTTGTCTGTC GCAATAAGGAATGGCGGGAAGCAACATTCATTTCACCTTATAATGCTATGAACCAGAGAGCCTACCGTATGCTT- GGACTTAATGTTCAGACAG TAGACTCGTCTCAAGGTTCGGAGTATGATTATGTTATCTTTTGTGTTACTGCAGATTCGCAGCATGCACTGAAT- ATTAACAGATTCAATGTAG CGCTTACAAGAGCCAAGCGTGGTATACTAGTTGTCATGCGTCAGCGTGATGAACTATATTCAGCTCTTAAGTTT- ATAGAGCTTGATAGTGTAG CAAGTCTGCAAGGTACAGGCTTGTTTAAAATTTGCAACAAAGAGTTTAGTGGTGTTCACCCAGCTTATGCAGTC- ACAACTAAGGCTCTTGCTG CAACTTATAAAGTTAATGATGAACTTGCTGCACTTGTTAACGTGGAAGCTGGTTCAGAAATAACATATAAACAT- CTTATTTCTTTGTTAGGGT TTAAGATGAGTGTTAATGTTGAAGGCTGCCACAACATGTTTATAACACGTGATGAGGCTATCCGCAACGTAAGA- GGTTGGGTAGGTTTTGATG TAGAAGCAACACATGCTTGCGGTACTAACATTGGTACTAACCTGCCTTTCCAAGTAGGTTTCTCTACTGGTGCA- GACTTTGTAGTTACGCCTG AGGGACTTGTAGATACTTCAATAGGCAATAATTTTGAGCCTGTGAATTCTAAAGCACCTCCAGGTGAACAATTT- AATCACTTGAGAGCGTTAT TCAAAAGTGCTAAACCTTGGCATGTTGTAAGGCCAAGGATTGTGCAAATGTTAGCGGATAACCTGTGCAACGTT- TCAGATTGTGTAGTGTTTG TCACGTGGTGTCATGGCCTAGAACTAACCACTTTGCGCTATTTTGTTAAAATAGGCAAGGACCAAGTTTGTTCT- TGCGGTTCTAGAGCAACAA CTTTTAATTCTCATACTCAGGCTTATGCTTGTTGGAAGCATTGCTTGGGTTTTGATTTTGTTTATAATCCACTC- TTAGTGGATATTCAACAGT GGGGTTATTCTGGTAACCTACAATTTAACCATGATTTGCATTGTAATGTGCATGGACACGCACATGTAGCTTCT- GCGGATGCTATTATGACGC GTTGTCTTGCAATTAATAATGCATTTTGTCAAGATGTCAACTGGGATTTAACTTACCCTCATATAGCAAATGAG- GATGAAGTCAATTCTAGCT GTAGATATTTACAACGCATGTATCTTAATGCATGTGTTGATGCTCTTAAAGTTAACGTTGTCTATGATATAGGC- AACCCTAAAGGTATAAAAT GTGTTAGACGTGGAGACTTAAATTTTAGATTCTATGATAAGAATCCAATAGTACCCAATGTCAAGCAGTTTGAG- TATGACTATAATCAGCACA AAGATAAGTTTGCTGATGGTCTTTGTATGTTTTGGAATTGTAATGTGGATTGTTATCCCGACAATTCCTTAGTT- TGTAGGTACGACACACGAA ATTTGAGTGTGTTTAACCTACCTGGTTGTAATGGTGGTAGCTTGTATGTTAACAAGCATGCATTCCACACACCT- AAATTTGATCGCACTAGCT TTCGTAATTTGAAAGCTATGCCATTCTTTTTCTATGACTCATCGCCTTGCGAGACCATTCAATTGGATGGAGTT- GCGCAAGACCTTGTGTCAT TAGCTACGAAAGATTGTATCACAAAATGCAACATAGGCGGTGCTGTTTGTAAAAAGCACGCACAAATGTATGCA- GATTTTGTGACTTCTTATA ATGCAGCTGTTACTGCTGGTTTTACTTTTTGGGTTACTAATAATTTTAACCCATATAATTTGTGGAAAAGTTTT- TCAGCTCTCCAGTCTATCG ACAATATTGCTTATAATATGTATAAGGGTGGTCATTATGATGCTATTGCAGGAGAAATGCCCACTATCGTAACT- GGAGATAAAGTTTTTGTTA TAGATCAAGGCGTAGAAAAAGCAGTTTTTTTTAATCAAACAATTCTGCCTACATCTGTAGCGTTTGAGCTGTAT- GCGAAGAGAAATATTCGCA CACTGCCAAACAACCGTATTTTGAAAGGTTTGGGTGTAGATGTGACTAATGGATTTGTAATTTGGGATTACACG- AACCAAACACCACTATACC GTAATACTGTTAAGGTATGTGCATATACAGACATAGAACCAAATGGCCTAATAGTGCTGTATGATGATAGATAT- GGTGATTACCAGTCTTTTC TAGCTGCTGATAATGCTGTTTTAGTTTCTACACAGTGTTACAAGCGGTATTCGTATGTAGAAATACCGTCAAAC- CTGCTTGTTCAGAACGGTA TTCCGTTAAAAGATGGAGCGAACCTGTATGTTTATAAGCGTGTTAATGGTGCGTTTGTTACGCTACCTAACACA- TTAAACACACAGGGTCGCA GTTATGAAACTTTTGAACCTCGTAGTGATGTTGAGCGTGATTTTCTCGACATGTCTGAGGAGAGTTTTGTAGAA- AAGTATGGTAAAGAATTAG GTCTACAGCACATACTGTATGGTGAAGTTGATAAGCCCCAATTAGGTGGTTTACACACTGTTATAGGTATGTGC- AGACTTTTACGTGCGAATA AGTTGAACGCAAAGTCTGTTACTAATTCTGATTCTGATGTCATGCAAAATTATTTTGTATTGGCAGACAATGGT- TCCTACAAGCAAGTGTGTA CTGTTGTGGATTTGCTGCTTGATGATTTCTTAGAACTTCTTAGGAACATACTGAAAGAGTATGGTACTAATAAG- TCTAAAGTTGTAACAGTGT CAATTGATTACCATAGCATAAATTTTATGACTTGGTTTGAAGATGGCATTATTAAAACATGTTATCCACAGCTT- CAATCAGCATGGACGTGTG GTTATAATATGCCTGAACTTTATAAAGTTCAGAATTGTGTTATGGAACCTTGCAACATTCCTAATTATGGTGTT- GGAATAGCGTTGCCAAGTG GTATTATGATGAATGTGGCAAAGTATACACAACTCTGTCAATACCTTTCGAAAACAACAATGTGTGTACCGCAT- AATATGCGAGTAATGCATT TTGGAGCTGGAAGTGACAAAGGAGTGGCTCCAGGTAGTACTGTTCTTAAACAATGGCTCCCAGAAGGGACACTC- CTTGTCGATAATGATATTG TAGACTATGTGTCTGATGCACATGTTTCTGTGCTTTCAGATTGCAATAAATATAAGACAGAGCACAAGTTTGAT- CTTGTGATATCTGATATGT ATACAGACAATGATTCAAAAAGAAAGCATGAAGGCGTGATAGCCAATAATGGCAATGATGACGTTTTCATATAT- CTCTCAAGTTTTCTTCGTA ATAATTTGGCTCTAGGTGGTAGTTTTGCTGTAAAAGTGACAGAGACAAGTTGGCACGAAGTTTTATATGACATT- GCACAGGATTGTGCATGGT GGACAATGTTTTGTACAGCAGTGAATGCCTCTTCTTCAGAAGCATTCTTGGTTGGTGTTAATTATTTGGGTGCA- AGTGAAAAGGTTAAGGTTA GTGGAAAAACGCTGCACGCAAATTATATATTTTGGAGGAATTGTAATTATTTACAAACCTCTGCTTATAGTATA- TTTGACGTTGCTAAGTTTG ATTTGAGATTGAAAGCAACACCAGTTGTTAATTTGAAAACTGAACAAAAGACAGACTTAGTCTTTAATTTAATT- AAGTGTGGTAAGTTACTGG TAAGAGATGTTGGTAACACCTCTTTTACTAGTGACTCTTTTGTGTGTACTATGTAGTGCTGCTTTGTATGACAG- TAGTTCTTACGTTTACTAC TACCAAAGTGCCTTTAGACCACCTAATGGTTGGCATTTACACGGGGGTGCTTATGCGGTAGTTAATATTTCTAG- CGAATCTAATAATGCAGGC TCTTCACCTGGGTGTATTGTTGGTACTATTCATGGTGGTCGTGTTGTTAATGCTTCTTCTATAGCTATGACGGC- ACCGTCATCAGGTATGGCT TGGTCTAGCAGTCAGTTTTGTACTGCACACTGTAACTTTTCAGATACTACAGTGTTTGTTACACATTGTTATAA- ATATGATGGGTGTCCTATA ACTGGCATGCTTCAAAAGAATTTTTTACGTGTTTCTGCTATGAAAAATGGCCAGCTTTTCTATAATTTAACAGT- TAGTGTAGCTAAGTACCCT ACTTTTAAATCATTTCAGTGTGTTAATAATTTAACATCCGTATATTTAAATGGTGATCTTGTTTACACCTCTAA- TGAGACCACAGATGTTACA TCTGCAGGTGTTTATTTTAAAGCTGGTGGACCTATAACTTATAAAGTTATGAGAGAAGTTAAAGCCCTGGCTTA- TTTTGTTAATGGTACTGCA CAAGATGTTATTTTGTGTGATGGATCACCTAGAGGCTTGTTAGCATGCCAGTATAATACTGGCAATTTTTCAGA- TGGCTTTTATCCTTTTATT AATAGTAGTTTAGTTAAGCAGAAGTTTATTGTCTATCGTGAAAATAGTGTTAATACTACTTTTACGTTACACAA- TTTCACTTTTCATAATGAG ACTGGCGCCAACCCTAATCCTAGTGGTGTTCAGAATATTCAAACTTACCAAACACAAACAGCTCAGAGTGGTTA- TTATAATTTTAATTTTTCC TTTCTGAGTAGTTTTGTTTATAAGGAGTCTAATTTTATGTATGGATCTTATCACCCAAGTTGTAATTTTAGACT- AGAAACTATTAATAATGGC TTGTGGTTTAATTCACTTTCAGTTTCAATTGCTTACGGTCCTCTTCAAGGTGGTTGCAAGCAATCTGTCTTTAG- TGGTAGAGCAACTTGTTGT TATGCTTATTCATATGGAGGTCCTTCGCTGTGTAAAGGTGTTTATTCAGGTGAGTTAGATCTTAATTTTGAATG- TGGACTGTTAGTTTATGTT ACTAAGAGCGGTGGCTCTCGTATACAAACAGCCACTGAACCGCCAGTTATAACTCGACACAATTATAATAATAT- TACTTTAAATACTTGTGTT GATTATAATATATATGGCAGAACTGGCCAAGGTTTTATTACTAATGTAACCGACTCAGCTGTTAGTTATAATTA- TCTAGCAGACGCAGGTTTG GCTATTTTAGATACATCTGGTTCCATAGACATCTTTGTTGTACAAGGTGAATATGGTCTTACTTATTATAAGGT- TAACCCTTGCGAAGATGTC AACCAGCAGTTTGTAGTTTCTGGTGGTAAATTAGTAGGTATTCTTACTTCACGTAATGAGACTGGTTCTCAGCT- TCTTGAGAACCAGTTTTAC ATTAAAATCACTAATGGAACACGTCGTTTTAGACGTTCTATTACTGAAAATGTTGCAAATTGCCCTTATGTTAG- TTATGGTAAGTTTTGTATA AAACCTGATGGTTCAATTGCCACAATAGTACCAAAACAATTGGAACAGTTTGTGGCACCTTTACTTAATGTTAC- TGAAAATGTGCTCATACCT AACAGTTTTAATTTAACTGTTACAGATGAGTACATACAAACGCGTATGGATAAGGTCCAAATTAATTGTCTGCA- GTATGTTTGTGGCAATTCT CTGGATTGTAGAGATTTGTTTCAACAATATGGGCCTGTTTGTGACAACATATTGTCTGTAGTAAATAGTATTGG- TCAAAAAGAAGATATGGAA CTTTTGAATTTCTATTCTTCTACTAAACCGGCTGGTTTTAATACACCATTTCTTAGTAATGTTAGCACTGGTGA- GTTTAATATTTCTCTTCTG TTAACAACTCCTAGTAGTCCTAGAAGGCGTTCTTTTATTGAAGACCTTCTATTTACAAGCGTTGAATCTGTTGG- ATTACCAACAGATGACGCA TACAAAAATTGCACTGCAGGACCTTTAGGTTTTCTTAAGGACCTTGCGTGTGCTCGTGAATATAATGGTTTGCT- TGTGTTGCCTCCCATTATA ACAGCAGAAATGCAAATTTTGTATACTAGTTCTCTAGTAGCTTCTATGGCTTTTGGTGGTATTACTGCAGCTGG- TGCTATACCTTTTGCCACA CAACTGCAGGCTAGAATTAATCACTTGGGTATTACCCAGTCACTTTTGTTGAAGAATCAAGAAAAAATTGCTGC- TTCCTTTAATAAGGCCATT GGTCGTATGCAGGAAGGTTTTAGAAGTACATCTCTAGCATTACAACAAATTCAAGATGTTGTTAATAAGCAGAG- TGCTATTCTTACTGAGACT ATGGCATCACTTAATAAAAATTTTGGTGCTATTTCTTCTATGATTCAAGAAATCTACCAGCAACTTGACGCCAT- ACAAGCAAATGCTCAAGTG GATCGTCTTATAACTGGTAGATTGTCATCACTTTCTGTTTTAGCATCTGCTAAGCAGGCGGAGCATATTAGAGT- GTCACAACAGCGTGAGTTA GCTACTCAGAAAATTAATGAGTGTGTTAAGTCACAGTCTATTAGGTACTCCTTTTGTGGTAATGGACGACATGT- TCTAACCATACCGCAAAAT GCACCTAATGGTATAGTGTTTATACACTTTTCTTATACTCCAGATAGTTTTGTTAATGTTACTGCAATAGTGGG- TTTTTGTGTAAAGCCAGCT

AATGCTAGTCAGTATGCAATAGTACCCGCTAATGGTAGGGGTATTTTTATACAAGTTAATGGTAGTTACTACAT- CACAGCACGAGATATGTAT ATGCCAAGAGCTATTACTGCAGGAGATATAGTTACGCTTACTTCTTGTCAAGCAAATTATGTAAGTGTAAATAA- GACCGTCATTACTACATTC GTAGACAATGATGATTTTGATTTTAATGACGAATTGTCAAAATGGTGGAATGACACTAAGCATGAGCTACCAGA- CTTTGACAAATTCAATTAC ACAGTACCTATACTTGACATTGATAGTGAAATTGATCGTATTCAAGGCGTTATACAGGGTCTTAATGACTCTTT- AATAGACCTTGAAAAACTT TCAATACTCAAAACTTATATTAAGTGGCCTTGGTATGTGTGGTTAGCCATAGCTTTTGCCACTATTATCTTCAT- CTTAATACTAGGATGGGTT TTCTTCATGACTGGATGTTGTGGTTGTTGTTGTGGATGCTTTGGCATTATGCCTCTAATGAGTAAGTGTGGTAA- GAAATCTTCTTATTACACG ACTTTTGATAACGATGTGGTAACTTAACAATACAGACCTAAAAAGTCTGTTTAATGATTCAAAGTCCCACGTCC- TTCCTAATAGTATTAATTT TTCTTTGGTGTAAACTTGTACTAAGTTGTTTTAGAGAGTTTATTATAGCGCTCCAACAACTAATACAAGTTTTA- CTCCAAATTATCAATAGTA ACTTACAGCCTAGACTGACCCTTTGTCACAGTCTAGACTAATGTTAAACTTAGAAGCAATTATTGAAACTGGTG- AGCAAGTGATTCAAAAAAT CAGTTTCAATTTACAGCATATTTCAAGTGTATTAAACACAGAAGTATTTGACCCCTTTGACTATTGTTATTACA- GAGGAGGTAATTTTTGGGA AATAGAGTCAGCTGAAGATTGTTCAGGTGATGATGAATTTATTGAATAAGTCGCTAGAGGAAAATGGAAGTTTT- CTAACAGCGCTTTATATAT TTGTAGGATTTTTAGCACTTTATCTTCTAGGTAGAGCACTTCAAGCATTTGTACAGGCTGCTGATGCTTGTTGT- TTATTTTGGTATACATGGG TAGTAATTCCAGGAGCTAAGGGTACAGCCTTTGTATATAAGTATACATATGGTAGAAAACTTAACAATCCGGAA- TTAGAAGCAGTTATTGTCA ACGAGTTTCCTAAGAACGGTTGGAATAATAAAAATCCAGCAAATTTTCAAGATGTCCAACGAGACAAATTGTAC- TCTTGACTTTGAACAGTCA GTTGAGCTTTTTAAAGAGTATAATTTATTTATAACTGCATTCTTGTTGTTCTTAACCATAATACTTCAGTATGG- CTATGCAACAAGAAGTAAG TTTATTTATATACTGAAAATGATAGTGTTATGGTGCTTTTGGCCCCTTAACATTGCAGTAGGTGTAATTTCATG- TATATACCCACCAAACACA GGAGGTCTTGTCGCAGCGATAATACTTACAGTGTTTGCGTGTCTGTCTTTTGTAGGTTATTGGATCCAGAGTAT- TAGACTCTTTAAGCGGTGT AGGTCATGGTGGTCATTTAACCCAGAATCTAATGCCGTAGGTTCAATACTCCTAACTAATGGTCAACAATGTAA- TTTTGCTATAGAGAGTGTG CCAATGGTGCTTTCTCCAATTATAAAGAATGGTGTTCTTTATTGTGAGGGTCAGTGGCTTGCTAAGTGTGAACC- AGACCACTTGCCTAAAGAT ATATTTGTTTGTACACCGGATAGACGTAATATCTACCGTATGGTGCAGAAATATACTGGTGACCAAAGCGGAAA- TAAGAAACGGTTTGCTACG TTTGTCTATGCAAAGCAGTCAGTAGATACTGGCGAGCTAGAAAGTGTAGCAACAGGAGGGAGTAGTCTTTACAC- CTAAATGTGTGTGTGTAGA GAGTATTTAAAATTATTCTTTAATAGTGCCTCTATTTTAAGAGCGCATAATAGTATTATTTTTGAGGATATTAA- TATAAATCCTCTCTGTTTT ATACTCTCTTTTCAAGAGCTATTATTTAAAAAACAGTTTTTCCACTCTTTTGTGCCAAAAACTATTGTTGTTAA- TGGTGTAACCTTTCAAGTA GATAATGGAAAAGTCTACTACGAAGGAAAACCAATTTTTCAGAAAGGTTGTTGTAGGTTGTGGTTGAGTTATAA- AAAAGATTAAACTACCTAC TACACTTATTTTTATAAGAGGCGTTTTATCTTACAAGCGCTTAATAAATACGGACGATGAAATGGCTGACTAGT- TTTGTAAGGGCAGTTATTT CATGTTATAAACCCCTATTATTAACTCAATTAAGAGTATTAGATAGGTTAATCTTAGATCATGGACCAAAACAC- ATCTTAACGTGTGTTAGGT GCGTGATTTTGTTTCAATTAGATTTAGTTTATAGGTTGGCGTATACGCCTACTCAATCGCTGGTATGAATAATA- GTAAAGATAATCCTTTTTG CGGAGCAATAGCAAGAAAAGCGCGAATTTATCTGAGAGAAGGATTAGATTGTGTTTACTTTCTTAACAAAGCAG- GACAAGCAGAGTCTTGTCC CGCGTGTACCTCTCTAGTATTCCAGGGGAAAACTTGTGAGGAACACAAATATAATAATAATCTTTTGTCATGGC- AAGCGGTAAGGCAACTGGA AAGACAGATGCCCCAGCTCCAGTCATCAAACTAGGAGGACCAAAGCCACCTAAAGTTGGTTCTTCTGGAAATGT- ATCTTGGTTTCAAGCAATA AAAGCCAAGAAGTTAAATTCACCTCCGCCTAAGTTTGAAGGTAGCGGTGTTCCTGATAATGAAAATCTAAAACC- AAGTCAGCAGCATGGATAT TGGAGACGCCAAGCTAGGTTTAAGCCAGGTAAAGGTGGAAGAAAACCAGTCCCAGATGCTTGGTATTTTTACTA- TACTGGAACAGGACCAGCC GCTAACCTGAATTGGGGTGATAGCCAAGATGGTATAGTGTGGGTTGCTGGTAAGGGTGCTGATACTAAATTTAG- ATCTAATCAGGGTACTCGT GACTCTGACAAGTTTGACCAATATCCGCTACGGTTTTCAGACGGAGGACCTGATGGTAATTTCCGTTGGGATTT- CATTCCTCTGAATCGTGGC AGGAGTGGGAGATCAACAGCAGCTTCATCAGCAGCATCTAGTAGAGCACCATCACGTGAAGTTTCGCGTGGTCG- CAGGAGTGGTTCTGAAGAT GATCTTATTGCTCGTGCAGCAAGGATAATTCAGGATCAGCAGAAGAAGGGTTCTCGCATTACAAAGGCTAAGGC- TGATGAAATGGCTCACCGC CGGTATTGCAAGCGCACTATTCCACCTAATTATAAGGTTGATCAAGTGTTTGGTCCCCGTACTAAAGGTAAGGA- GGGAAATTTTGGTGATGAC AAGATGAATGAGGAAGGTATTAAGGATGGGCGCGTTACAGCAATGCTCAACCTAGTTCCTAGCAGCCATGCTTG- TCTTTTCGGAAGTAGAGTG ACGCCCAGACTTCAACCAGATGGGCTGCACTTGAAATTTGAATTTACTACTGTGGTCCCACGTGATGATCCGCA- GTTTGATAATTATGTAAAA ATTTGTGATCAGTGTGTTGATGGTGTAGGAACACGTCCAAAAGATGATGAACCAAGACCAAAGTCACGCTCAAG- TTCAAGACCTGCAACAAGA GGAAATTCTCCAGCGCCAAGACAGCAGCGCCCTAAGAAGGAGAAAAAGCCAAAGAAGCAGGATGATGAAGTGGA- TAAAGCATTGACCTCAGAT GAGGAGAGGAACAATGCACAGCTGGAATTTGATGATGAACCCAAGGTAATTAACTGGGGGGATTCAGCCCTAGG- AGAGAATGAACTTTGAGTA AAATTCAATAGTAAGAGTTAAGGAAGATAGGCATGTAGCTTGATTACCTACATGTCTATCGCCAGGGAAATGTC- TAATTTGTCTACTTAGTAG CCTGGAAACGAACGGTAGACCCTTAGATTTTAATTTAGTTTAATTTTTAGTTTAGTTTAAGTTAGTTTAGAGTA- GGTATAAAGATGCCAGTGC CGGGGCCACGCGGAGTACGACCGAGGGTACAGCACTAGGACGCCCATTAGGGGAAGAGCTAAATTTTAGTTTAA- GTTAAGTTTAATTGGCTAT GTATAGTTAAAATTTATAGGCTAGTATAGAGTTAGAGCAAAAAAAAAAAAAAAAAAAAAAAAAAA

Accessory Proteins

[0107] 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.

[0108] 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.

TABLE-US-00002 (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

[0109] Replicase

[0110] 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.

[0111] Thus Gene 1 in IBV encodes 15 (16 in other coronaviruses) non-structural proteins (nsp2-16), which are associated with RNA replication and transcription.

[0112] The term `replicase protein` is used herein to refer to the pp1a and pp1ab polyproteins or individual nsp subunits.

[0113] The term `replicase gene` is used herein to refer to a nucleic acid sequence which encodes for replicase proteins.

[0114] A summary of the functions of coronavirus nsp proteins is provided in Table 1.

TABLE-US-00003 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

[0115] 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.

[0116] The nucleotide and protein sequences for nsp-3 in IBV M41 are provided as SEQ ID NOs: 5 and 6, respectively.

TABLE-US-00004 (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

[0117] Reduced Pathogenicity

[0118] 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.

[0119] 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.

[0120] 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.

[0121] 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.

[0122] 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.

[0123] 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.

[0124] 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.

[0125] The attenuated coronavirus of the present invention may cause a reduced level of clinical symptoms compared to the corresponding wild-type coronavirus.

[0126] 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.

[0127] 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.

[0128] 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.

[0129] 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-US-00005 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.

[0130] The attenuated coronavirus of the present invention may replicate at non-pathogenic levels in ovo.

[0131] 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.

[0132] 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.

[0133] 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.

[0134] 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.

[0135] 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).

[0136] 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.

[0137] 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.

[0138] Variant

[0139] 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.

[0140] 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.

[0141] 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).

[0142] 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).

[0143] 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.

[0144] 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:

TABLE-US-00006 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

[0145] 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.

[0146] 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.

[0147] 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.

[0148] The mutation in nsp-3 may comprise one or more amino acid mutations selected from the list of:

[0149] a) Asn (N) to Ala (A) at position 373 in SEQ ID NO: 6; and

[0150] b) Gly (G) to Ser (S) at position 379 in SEQ ID NO: 6.

[0151] The mutation in nsp-3 may comprise one or more nucleotide substitutions which encodes an amino acid mutation as described herein.

[0152] The mutation in nsp-3 may comprise one or more nucleotide substitutions selected from the list of:

[0153] 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

[0154] b) G to A at nucleotide position 1138 compared to the sequence shown as SEQ ID NO: 5.

[0155] 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.

[0156] The mutation in nsp-3 may comprise the amino acid mutation Asn (N) to Ala (A) at position 373 in SEQ ID NO: 3.

[0157] The mutation in nsp-3 may comprise the amino acid mutation Gly (G) to Ser (S) at position 379 in SEQ ID NO: 3.

[0158] 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.

[0159] The attenuated coronavirus may also be defined at the nucleotide level.

[0160] For example the nucleotide sequence of the attenuated coronavirus of the present invention may comprise one or more mutations selected from the list of:

[0161] 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

[0162] b) G to A at nucleotide position 1138 compared to the sequence shown as SEQ ID NO: 5.

[0163] 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.

[0164] 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.

[0165] 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.

[0166] 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.

[0167] 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.

[0168] 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:

[0169] The nucleotide sequence may comprise any combination of the nucleotide mutations listed above.

[0170] 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.

[0171] 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.

[0172] For example the nucleotide sequence of the attenuated coronavirus of the present invention may comprise the following mutations:

[0173] 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

[0174] b) G to A at nucleotide position 1138 compared to the sequence shown as SEQ ID NO: 5; and

[0175] c) deletion from nucleotide position 37 to 384 of the gene 3 sequence shown as SEQ ID NO: 2.

[0176] The nucleotide sequence may not comprise a substitution which corresponds to the C12008T substitution reported by Ammayappan et al. (as above).

[0177] 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.

[0178] The nucleotide sequence may be codon optimised for production in the host/host cell of choice.

[0179] It may be isolated, or as part of a plasmid, virus or host cell.

[0180] Plasmid

[0181] 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.

[0182] 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.

[0183] 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.

[0184] 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.

[0185] Recombinant Vaccinia Virus

[0186] The present invention also relates to a recombinant vaccinia virus (rVV) comprising a variant gene as defined herein.

[0187] The recombinant vaccinia virus (rVV) may be made using a vaccinia-virus based reverse genetics system.

[0188] In this respect, the present invention also provides a method for making a viral particle by:

[0189] (i) transfecting a plasmid as described in the previous section into a host cell;

[0190] (ii) infecting the host cell with a recombining virus comprising the genome of a coronavirus strain with a nsp-3 gene or gene 3;

[0191] (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;

[0192] (iv) selecting for recombining virus comprising the modified gene.

[0193] 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

[0194] 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.

[0195] 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.

[0196] 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.

[0197] The above method optionally includes the step:

[0198] (v) recovery of recombinant coronavirus comprising the modified gene from the DNA from the recombining virus from step (iv).

[0199] 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).

[0200] 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.

[0201] 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.

[0202] Positive recombinants may then be verified to contain the modified gene by, for example, PCR and sequencing.

[0203] 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)).

[0204] 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).

[0205] Cell

[0206] The coronavirus may be used to infect a cell.

[0207] Coronavirus particles may be harvested, for example from the supernatant, by methods known in the art, and optionally purified.

[0208] The cell may be used to produce the coronavirus particle.

[0209] Thus the present invention also provides a method for producing a coronavirus which comprises the following steps:

[0210] (i) infection of a cell with a coronavirus according to the invention;

[0211] (ii) allowing the virus to replicate in the cell; and

[0212] (iii) harvesting the progeny virus.

[0213] 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.

[0214] The cell may be able to produce recombinant recombining virus (e.g. vaccinia virus) containing the mutation(s).

[0215] 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.

[0216] Vaccine

[0217] 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.

[0218] 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.

[0219] 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.

[0220] Vaccination Method

[0221] The coronavirus of the present invention may be used to treat and/or prevent a disease.

[0222] 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.

[0223] 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.

[0224] 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.

[0225] 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.

[0226] The disease may be infectious bronchitis.

[0227] 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.

[0228] The vaccine may be provided in a form suitable for its administration, such as an eye-dropper for intra-ocular use.

[0229] 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.

[0230] 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.

[0231] 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.

[0232] The process can be automated using a robotic injection process, such as those described in WO 2004/078203.

[0233] 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.

[0234] 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).

[0235] 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).

[0236] 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.

[0237] 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.

[0238] 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.

[0239] 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.

[0240] 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.

[0241] 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.

[0242] 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.

[0243] 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".

[0244] 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

[0245] 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.

[0246] 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

[0247] 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.

[0248] 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).

[0249] 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.

[0250] 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.

[0251] 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.

[0252] 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

[0253] 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 10.sup.3.65 EID.sub.50 per dose.

[0254] 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-US-00007 TABLE 3 Between hatch and End of Treatment Eggs At hatch challenge Challenge study Saline MDA.sup.- Determine Clinical Chickens 5 days post IB M41- MDA.sup.- hatch examination at age of challenge a K-A- percent- of birds 21 days ciliostasis ADRP ages test will be IB M41- MDA.sup.- done K-S- ADRP IB M41- MDA.sup.- 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.

[0255] Results of Hatchability Study

[0256] SPF broiler eggs were incubated according to routine incubation procedures. At 19 days incubation 6 groups of 30 eggs were inoculated with 10.sup.4 EID.sup.50 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:

[0257] PBS 87% (26/30) hatched, 1 chick died after hatching and 3 eggs remained unopened and unmarked for embryo injection.

[0258] M41-K-A-ARDP 97% (29/30) hatched, 1 egg remained unopened and unmarked for embryo injection.

[0259] 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.

[0260] 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.

[0261] 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.

[0262] 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.

Sequence CWU 1

1

6127500DNAAvian infectious bronchitis virus 1acttaagata gatattaata tatatctatc acactagcct tgcgctagat ttccaactta 60acaaaacgga cttaaatacc tacagctggt cctcataggt gttccattgc agtgcacttt 120agtgccctgg atggcacctg gccacctgtc aggtttttgt tattaaaatc ttattgttgc 180tggtatcact gcttgttttg ccgtgtctca ctttatacat ccgttgcttg ggctacctag 240tatccagcgt cctacgggcg ccgtggctgg ttcgagtgcg aagaacctct ggttcatcta 300gcggtaggcg ggtgtgtgga agtagcactt cagacgtacc ggttctgttg tgtgaaatac 360ggggtcacct ccccccacat acctctaagg gcttttgagc ctagcgttgg gctacgttct 420cgcataaggt cggctatacg acgtttgtag ggggtagtgc caaacaaccc ctgaggtgac 480aggttctggt ggtgtttagt gagcagacat acaatagaca gtgacaacat ggcttcaagc 540ctaaaacagg gagtatctcc caaactaagg gatgtcattc ttgtatccaa agacattcct 600gaacaacttt gtgacgcttt gtttttctat acgtcacaca accctaagga ttacgctgat 660gcttttgcag ttaggcagaa gtttgatcgt aatctgcaga ctgggaaaca gttcaaattt 720gaaactgtgt gtggtctctt cctcttgaag ggagttgaca aaataacacc tggcgtccca 780gcaaaagtct taaaagccac ttctaagttg gcagatttag aagacatctt tggtgtctct 840ccctttgcaa gaaaatatcg tgaacttttg aagacagcat gccagtggtc tcttactgta 900gaaacactgg atgctcgtgc acaaactctt gatgaaattt ttgaccctac tgaaatactt 960tggcttcagg tggcagcaaa aatccaagtt tcggctatgg cgatgcgcag gcttgttgga 1020gaagtaactg caaaagtcat ggatgctttg ggctcaaata tgagtgctct tttccagatt 1080tttaaacaac aaatagtcag aatttttcaa aaagcgctgg ctatttttga gaatgtgagt 1140gaattaccac agcgtattgc agcacttaag atggcttttg ctaagtgtgc caagtccatt 1200actgttgtgg ttatggagag gactctagtt gttagagagt tcgcaggaac ttgtcttgca 1260agcattaatg gtgctgttgc aaaattcttt gaagaactcc caaatggttt catgggtgct 1320aaaattttca ctacacttgc cttctttagg gaggctgcag tgaaaattgt ggataacata 1380ccaaatgcac cgagaggcac taaagggttt gaagtcgttg gtaatgccaa aggtacacaa 1440gttgttgtgc gtggcatgcg aaatgactta acactgcttg accaaaaagc tgaaattcct 1500gtggagtcag aaggttggtc tgcaattttg ggtggacatc tttgctatgt ctttaagagt 1560ggtgatcgct tttacgcggc acctctttca ggaaattttg cattgcatga tgtgcattgt 1620tgtgagcgtg ttgtctgtct ttctgatggt gtaacaccgg agataaatga tggacttatt 1680cttgcagcaa tctactcttc ttttagtgtc gcagaacttg tggcagccat taaaaggggt 1740gaaccattta agtttctggg tcataaattt gtgtatgcaa aggatgcagc agtttctttt 1800acattagcga aggctgctac tattgcagat gttttgaagc tgtttcaatc agcgcgtgtg 1860aaagtagaag atgtttggtc ttcacttact gaaaagtctt ttgaattctg gaggcttgca 1920tatggaaaag tgcgtaatct cgaagaattt gttaagactt gtttttgtaa ggctcaaatg 1980gcgattgtga ttttagcgac agtgcttgga gagggcattt ggcatcttgt ttcgcaagtc 2040atctataaag taggtggtct ttttactaaa gttgttgact tttgtgaaaa atattggaaa 2100ggtttttgtg cacagttgaa aagagctaag ctcattgtca ctgaaaccct ctgtgttttg 2160aaaggagttg cacagcattg ttttcaacta ttgctggatg caatacagtt tatgtataaa 2220agttttaaga agtgtgcact tggtagaatc catggagact tgctcttctg gaaaggaggt 2280gtgcacaaaa ttattcaaga gggcgatgaa atttggtttg acgccattga tagtattgat 2340gttgaagatc tgggtgttgt tcaagaaaaa ttgattgatt ttgatgtttg tgataatgtg 2400acacttccag agaaccaacc cggtcatatg gttcaaatcg aggatgacgg aaagaactac 2460atgttcttcc gcttcaaaaa ggatgagaac atttattata caccaatgtc acagcttggt 2520gctattaatg tggtttgcaa agcaggcggt aaaactgtca cctttggaga aactactgtg 2580caagaaatac caccacctga tgttgtgttt attaaggtta gcattgagtg ttgtggtgaa 2640ccatggaata caatcttcaa aaaggcttat aaggagccca ttgaagtaga gacagacctc 2700acagttgaac aattgctctc tgtggtctat gagaaaatgt gtgatgatct caagctgttt 2760ccggaggctc cagaaccacc accatttgag aatgtcacac ttgttgataa gaatggtaaa 2820gatttggatt gcataaaatc atgccatctg atctatcgtg attatgagag cgatgatgac 2880atcgaggaag aagatgcaga agaatgtgac acggattcag gtgatgctga ggagtgtgac 2940actaattcag aatgtgaaga agaagatgag gatactaaag tgttggctct tatacaagac 3000ccggcaagta acaaatatcc tctgcctctt gatgatgatt atagcgtcta caatggatgt 3060attgttcata aggacgctct cgatgttgtg aatttaccat ctggtgaaga aacctttgtt 3120gtcaataact gctttgaagg ggctgttaaa gctcttccgc agaaagttat tgatgttcta 3180ggtgactggg gtgaggctgt tgatgcgcaa gaacaattgt gtcaacaaga atcaactcgg 3240gtcatatctg agaaatcagt tgagggtttt actggtagtt gtgatgcaat ggctgaacaa 3300gctattgttg aagagcagga aatagtacct gttgttgaac aaagtcagga tgtagttgtt 3360tttacacctg cagacctaga agttgttaaa gaaacagcag aagaggttga tgagtttatt 3420ctcatttctg ctgtccctaa agaagaagtt gtgtctcagg agaaagagga gccacaggtt 3480gagcaagagc ctaccctagt tgttaaagca caacgtgaga agaaggctaa aaagttcaaa 3540gttaaaccag ctacatgtga aaaacccaaa tttttggagt acaaaacatg tgtgggtgat 3600ttggctgttg taattgccaa agcattggat gagtttaaag agttctgcat tgtaaacgct 3660gcaaatgagc acatgtcgca tggtggtggc gttgcaaagg caattgcaga cttttgtgga 3720ccggactttg ttgaatattg cgcggactat gttaagaaac atggtccaca gcaaaaactt 3780gtcacacctt catttgttaa aggcattcaa tgtgtgaata atgttgtagg acctcgccat 3840ggagacagca acttgcgtga gaagcttgtt gctgcttaca agagtgttct tgtaggtgga 3900gtggttaact atgttgtgcc agttctctca tcagggattt ttggtgtaga ttttaaaata 3960tcaatagatg ctatgcgcga agcttttaaa ggttgtgcca tacgcgttct tttattttct 4020ctgagtcaag aacacatcga ttatttcgat gcaacttgta agcagaagac aatttatctt 4080acggaggatg gtgttaaata ccgctctgtt gttttaaaac ctggtgattc tttgggtcaa 4140tttggacagg tttttgcaag aaataaggta gtcttttcgg ctgatgatgt tgaggataaa 4200gaaatcctct ttatacccac aactgacaag actattcttg aatattatgg tttagatgcg 4260caaaagtatg taacatattt gcaaacgctt gcgcagaaat gggatgttca atatagagac 4320aattttgtta tattagagtg gcgtgacgga aattgctgga ttagttcagc aatagttctc 4380cttcaagctg ctaaaattag atttaaaggt tttcttgcag aagcatgggc taaactgttg 4440ggtggagatc ctacagactt tgttgcctgg tgttatgcaa gttgcaatgc taaagtaggt 4500gatttttcag atgctaattg gcttttggcc aatttagcag aacattttga cgcagattac 4560acaaatgcac ttcttaagaa gtgtgtgtcg tgcaattgtg gtgttaagag ttatgaactt 4620aggggtcttg aagcctgtat tcagccagtt cgagcaccta atcttctaca ttttaaaacg 4680caatattcaa attgcccaac ctgtggtgca agtagtacgg atgaagtaat agaagcttca 4740ttaccgtact tattgctttt tgctactgat ggtcctgcta cagttgattg tgatgaaaat 4800gctgtaggga ctgttgtttt cattggctct actaatagtg gccattgtta tacacaagcc 4860gatggtaagg cttttgacaa tcttgctaag gatagaaaat ttggaaggaa gtcgccttac 4920attacagcaa tgtatacacg tttttctctt aggagtgaaa atcccctact tgttgttgaa 4980catagtaagg gtaaagctaa agtagtaaaa gaagatgttt ctaaccttgc tactagttct 5040aaagccagtt ttgacgatct tactgacttt gaacagtggt atgatagcaa catctatgag 5100agtcttaaag tgcaggagac acctgataat cttgatgaat atgtgtcatt tacgacaaag 5160gaagattcta agttgccact gacacttaaa gttagaggta tcaaatcagt tgttgacttt 5220aggtctaagg atggttttac ttataagtta acacctgata ctgatgaaaa ttcaaaaaca 5280ccagtctact acccagtctt ggattctatt agtcttaggg caatatgggt tgaaggcagt 5340gctaattttg ttgttgggca tccaaattat tatagtaagt ctctccgaat tcccacgttt 5400tgggaaaatg ccgagagctt tgttaaaatg ggttataaaa ttgatggtgt aactatgggc 5460ctttggcgtg cagaacacct taataaacct aatttggaga gaatttttaa cattgctaag 5520aaagctattg ttggatctag tgttgttact acgcagtgtg gtaaaatact agttaaagca 5580gctacatacg ttgccgataa agtaggtgat ggtgtagttc gcaatattac agatagaatt 5640aagggtcttt gtggattcac acgtggccat tttgaaaaga aaatgtccct acaatttcta 5700aagacacttg tgttcttttt cttttatttc ttaaaggcta gtgctaagag tttagtttct 5760agctataaga ttgtgttatg taaggtggtg tttgctacct tacttatagt gtggtttata 5820tacacaagta atccagtagt gtttactgga atacgtgtgc tagacttcct atttgaaggt 5880tctttatgtg gtccttataa tgactacggt aaagattctt ttgatgtgtt acgctattgt 5940gcaggtgatt ttacttgtcg tgtgtgttta catgatagag attcacttca tctgtacaaa 6000catgcttata gcgtagaaca aatttataag gatgcagctt ctggcattaa ctttaattgg 6060aattggcttt atttggtctt tctaatatta tttgttaagc cagtggcagg ttttgttatt 6120atttgttatt gtgttaagta tttggtattg agttcaactg tgttgcaaac tggtgtaggt 6180tttctagatt ggtttgtaaa aacagttttt acccatttta attttatggg agcgggattt 6240tatttctggc tcttttacaa gatatacgta caagtgcatc atatattgta ctgtaaggat 6300gtaacatgtg aagtgtgcaa gagagttgca cgcagcaaca ggcaagaggt tagcgttgta 6360gttggtggac gcaagcaaat agtgcatgtt tacactaatt ctggctataa cttttgtaag 6420agacataatt ggtattgtag aaattgtgat gattatggtc accaaaatac atttatgtcc 6480cctgaagttg ctggcgagct ttctgaaaag cttaagcgcc atgttaaacc tacagcatat 6540gcttaccacg ttgtgtatga ggcatgcgtg gttgatgatt ttgttaattt aaaatataag 6600gctgcaattc ctggtaagga taatgcatct tctgctgtta agtgtttcag tgttacagat 6660tttttaaaga aagctgtttt tcttaaggag gcattgaaat gtgaacaaat atctaatgat 6720ggttttatag tgtgtaatac acagagtgcg catgcactag aggaagcaaa gaatgcagcc 6780gtctattatg cgcaatatct gtgtaagcca atacttatac ttgaccaggc actttatgag 6840caattaatag tagagcctgt gtctaagagt gttatagata aagtgtgtag cattttgtct 6900aatataatat ctgtagatac tgcagcttta aattataagg caggcacact tcgtgatgct 6960ctgctttcta ttactaaaga cgaagaagcc gtagatatgg ctatcttctg ccacaatcat 7020gaagtggaat acactggtga cggttttact aatgtgatac cgtcatatgg tatggacact 7080gataagttga cacctcgtga tagagggttt ttgataaatg cagatgcttc tattgctaat 7140ttaagagtca aaaatgctcc tccggtagta tggaagtttt ctgatcttat taaattgtct 7200gacagttgcc ttaaatattt aatttcagct actgtcaagt caggaggtcg tttctttata 7260acaaagtctg gtgctaaaca agttatttct tgtcataccc agaaactgtt ggtagagaaa 7320aaggcaggtg gtgttattaa taacactttt aaatggttta tgagttgttt taaatggctt 7380tttgtctttt atatactttt tacagcatgt tgtttgggtt actactatat ggagatgaat 7440aaaagttttg ttcaccccat gtatgatgta aactccacac tgcatgttga agggttcaaa 7500gttatagaca aaggtgttat tagagagatt gtgtcagaag ataattgttt ctctaataag 7560tttgttaatt ttgacgcctt ttggggtaaa tcatatgaaa ataataaaaa ctgtccaatt 7620gttacagttg ttatagatgg tgacgggaca gtagctgttg gtgttcctgg ttttgtatca 7680tgggttatgg atggtgttat gtttgtgcat atgacacaga ctgatcgtag accttggtac 7740attcctacct ggtttaatag agaaattgtt ggttacactc aggattcaat tatcactgag 7800ggtagttttt atacatctat agcattattt tctgctagat gtttatattt aacagccagc 7860aatacacctc aattgtattg ttttaatggc gacaatgatg cacctggagc cttaccattt 7920ggtagtatta ttcctcatag agtatacttc caacctaatg gtgttaggct tatagttcca 7980caacaaatac tgcatacacc ctacatagtg aagtttgttt cagacagcta ttgtagaggt 8040agtgtatgtg agtatactaa accaggttac tgtgtgtcac tagactccca atgggttttg 8100tttaatgatg aatacattag taaacctggc gttttctgtg gttctactgt tagagaactt 8160atgtttaata tggttagtac attctttact ggtgtcaacc ctaatattta tattcagcta 8220gcaactatgt ttttaatact agttgttatt gtgttaattt ttgcaatggt tataaagttt 8280caaggtgttt ttaaagctta tgcgaccatt gtgtttacaa taatgttagt ttgggttatt 8340aatgcatttg ttttgtgtgt acatagttat aatagtgttt tagctgttat attattagta 8400ctctattgct atgcatcatt ggttacaagt cgcaatactg ctataataat gcattgttgg 8460cttgttttta cctttggttt aatagtaccc acatggttgg cttgttgcta tctgggattt 8520attctttata tgtacacacc gttggttttc tggtgttacg gtactactaa aaatactcgt 8580aagttgtatg atggcaacga gtttgttggt aattatgacc ttgctgcgaa gagcactttt 8640gttattcgtg gtactgaatt tgttaagctt acgaatgaga taggtgataa atttgaagcc 8700tatctttctg cgtatgctag acttaaatac tattcaggca ctggtagtga gcaagattac 8760ttgcaagctt gtcgtgcatg gttagcttat gctttggacc aatatagaaa tagtggtgtt 8820gaggttgttt ataccccacc gcgttactct attggtgtta gtagactaca cgctggtttt 8880aaaaaactag tttctcctag tagtgctgtt gagaagtgca ttgttagtgt ctcttataga 8940ggcaataatc ttaatggact gtggctgggt gattctattt actgcccacg ccatgtgtta 9000ggtaagttta gtggtgacca gtggggtgac gtactaaacc ttgctaataa tcatgagttt 9060gaagttgtaa ctcaaaatgg tgttactttg aatgttgtca gcaggcggct taaaggagca 9120gttttaattt tacaaactgc agttgccaat gctgaaactc ctaagtataa gtttgttaaa 9180gctaattgtg gtgatagttt cactatagct tgttcttatg gtggtacagt tataggactt 9240taccctgtca ctatgcgttc taatggtact attagagcat ctttcctagc aggagcctgt 9300ggctcagttg gttttaatat agaaaagggt gtagttaatt tcttttatat gcaccatctt 9360gagttaccta atgcattaca cactggaact gacctaatgg gtgagtttta tggtggttat 9420gtagatgaag aggttgcgca aagagtgcca ccagataatc tagttactaa caatattgta 9480gcatggctct atgcggcaat tattagtgtt aaagaaagta gtttttcaca acctaaatgg 9540ttggagagta ctactgtttc tattgaagat tacaataggt gggctagtga taatggtttt 9600actccatttt ccactagtac tgctattact aaattaagtg ctataactgg ggttgatgtt 9660tgtaaactcc ttcgcactat tatggtaaaa agtgctcaat ggggtagtga tcccatttta 9720ggacaatata attttgaaga cgaattgaca ccagaatctg tatttaatca agttggtggt 9780gttaggttac agtcttcttt tgtaagaaaa gctacatctt ggttttggag tagatgtgta 9840ttagcttgct tcttgtttgt gttgtgtgct attgtcttat ttacggcagt gccacttaag 9900ttttatgtac atgcagctgt tattttgttg atggctgtgc tctttatttc ttttactgtt 9960aaacatgtta tggcatacat ggacactttc ctattgccta cattgattac agttattatt 10020ggagtttgtg ctgaagtccc tttcatatac aatactctaa ttagtcaagt tgttattttc 10080ttaagccaat ggtatgatcc tgtagtcttt gatactatgg taccatggat gttattgcca 10140ttagtgttgt acactgcttt taagtgtgta caaggctgct atatgaattc tttcaatact 10200tctttgttaa tgctgtatca gtttatgaag ttaggttttg ttatttacac ctcttcaaac 10260actcttactg catatacaga aggtaattgg gagttattct ttgagttggt tcacactatt 10320gtgttggcta atgttagtag taattcctta attggtttaa ttgtttttaa gtgtgctaag 10380tggattttat attattgcaa tgcaacatac tttaataatt atgtgttaat ggcagtcatg 10440gttaatggca taggctggct ttgcacctgt tactttggat tgtattggtg ggttaataaa 10500gtttttggtt taaccttagg taaatacaat tttaaagttt cagtagatca atataggtat 10560atgtgtttgc ataaggtaaa tccacctaaa actgtgtggg aggtctttac tacaaatata 10620cttatacaag gaattggagg cgatcgtgtg ttgcctatag ctacagtgca atctaaattg 10680agtgatgtaa agtgtacaac tgttgtttta atgcagcttt tgactaagct taatgttgaa 10740gcaaattcaa aaatgcatgc ttatcttgtt gagttacaca ataaaatcct cgcatctgat 10800gatgttggag agtgcatgga taatttattg ggtatgctta taacactatt ttgtatagat 10860tctactattg atttgggtga gtattgtgat gatatactta agaggtcaac tgtattacaa 10920tcggttactc aagagttttc gcacataccc tcgtatgctg aatatgaaag agctaagagt 10980atttatgaaa aggttttagc cgattctaaa aatggtggtg taacacagca agagcttgct 11040gcatatcgta aagctgccaa tattgcaaag tcagtttttg atagagactt ggctgttcaa 11100aagaagttag atagcatggc agaacgtgct atgacaacaa tgtataaaga ggcgcgtgta 11160actgatagaa gagcaaaatt agtttcatca ttacatgcac tacttttttc aatgcttaag 11220aaaatagatt ctgagaagct taatgtctta tttgaccagg cgaatagtgg tgttgtaccc 11280ctagcaactg ttccaattgt ttgtagtaat aagcttaccc ttgttatacc agacccagag 11340acgtgggtca agtgtgtgga gggtgtgcat gttacatatt caacagttgt ttggaatata 11400gactgtgtta ctgatgccga tggcacagag ttacacccca cttctacagg tagtggattg 11460acttactgta taagtggtga taatatagca tggcctttaa aggttaactt gactaggaat 11520gggcataata aggttgatgt tgccttgcaa aataatgagc ttatgcctca cggtgtaaag 11580acaaaggctt gcgtagcagg tgtagatcaa gcacattgta gcgttgagtc taaatgttat 11640tatacaagta ttagtggcag ttcagttgta gctgctatta cctcttcaaa tcctaatctg 11700aaagtagcct cttttttgaa tgaggcaggt aatcagattt atgtagactt agacccacca 11760tgtaaatttg gtatgaaagt gggtgataag gttgaagttg tttacctgta ttttataaaa 11820aatacgaggt ctattgtaag aggtatggta cttggtgcta tatctaatgt tgttgtgtta 11880caatctaaag gtcatgagac agaggaagtg gatgctgtag gcattctctc actttgttct 11940tttgcagtag atcctgcgga tacatattgt aaatatgtgg cagcaggtaa tcaaccttta 12000ggtaactgtg ttaaaatgtt gacagtacat aatggtagtg gttttgcaat aacatcaaag 12060ccaagtccaa ctccggatca ggattcttat ggaggagctt ctgtgtgtct ttattgtaga 12120gcacatatag cacaccctgg cggagcagga aatttagatg gacgctgtca atttaaaggt 12180tcttttgtgc aaatacctac tacggagaaa gatcctgttg gattctgtct acgtaacaag 12240gtttgcactg tttgtcagtg ttggattggt tatggatgtc agtgtgattc acttagacaa 12300cctaaacctt ctgttcagtc agttgctgtt gcatctggtt ttgataagaa ttatttaaac 12360gggtacgggg tagcagtgag gctcggctga tacccctagc taatggatgt gaccccgatg 12420ttgtaaagcg agcctttgat gtttgtaata aggaatcagc cggtatgttt caaaatttga 12480agcgtaactg tgcacgattc caagaagtac gtgatactga agatggaaat cttgagtatt 12540gtgattctta ttttgtggtt aaacaaacca ctcctagtaa ttatgaacat gagaaagctt 12600gttatgaaga cttaaagtca gaagtaacag ctgatcatga tttctttgtg ttcaataaga 12660acatttataa tattagtagg cagaggctta ctaagtatac tatgatggat ttttgctatg 12720ctttgcggca ctttgaccca aaggattgcg aagttcttaa agaaatactt gtcacttatg 12780gttgtataga agattatcac cctaagtggt ttgaagagaa taaggattgg tacgacccaa 12840tagaaaaccc taaatattat gccatgttgg ctaaaatggg acctattgta cgacgtgctt 12900tattgaatgc tattgagttc ggaaacctca tggttgaaaa aggttatgtt ggtgttatta 12960cacttgataa ccaagatctt aatggcaaat tttatgattt tggtgatttt cagaagacag 13020cgcctggtgc tggtgttcct gtttttgata cgtattattc ttacatgatg cccatcatag 13080ccatgactga tgcgttggca cctgagaggt attttgaata tgatgtgcat aagggttata 13140aatcttatga tctcctcaag tatgattata ctgaggagaa acaagatttg tttcagaagt 13200actttaagta ttgggatcaa gagtatcacc ctaactgtcg cgactgtagt gatgacaggt 13260gtttgataca ttgtgcaaac ttcaacatct tgttttctac acttgtaccg cagacttctt 13320tcggtaattt gtgtagaaag gtttttgttg atggtgtacc atttatagct acttgtggct 13380atcattctaa ggaacttggt gttattatga atcaagataa caccatgtca ttttcaaaaa 13440tgggtttgag tcaactcatg cagtttgttg gagatcctgc cttgttagtg gggacatcca 13500ataaattagt ggatcttaga acgtcttgtt ttagtgtttg tgctttagcg tctggtatta 13560ctcatcaaac ggtaaaacca ggtcacttta acaaggattt ctacgatttt gcagagaagg 13620ctggtatgtt taaggaaggt tcttctatac cacttaaaca tttcttctac ccacagactg 13680gtaatgctgc tataaacgat tatgattatt atcgttataa caggcctacc atgtttgata 13740tacgtcaact tttattttgt ttagaagtga cttctaaata ttttgaatgt tatgaaggcg 13800gctgtatacc agcaagccaa gttgtagtta acaatttaga taagagtgca ggttatccgt 13860tcaataagtt tggaaaggcc cgtctctatt atgaaatgag tctagaggag caggaccaac 13920tctttgagag tacaaagaag aacgtcctgc ctactataac tcagatgaat ttaaaatatg 13980ccatatccgc gaaaaataga gcgcgtacag tggcaggtgt gtctatcctt tctactatga 14040ctaataggca gtttcatcag aagattctta agtctatagt caacactaga aacgctcctg 14100tagttattgg aacaaccaag ttttatggcg gttgggataa catgttgaga aaccttattc 14160agggtgttga agacccgatt cttatgggtt gggattatcc aaagtgtgat agagcaatgc 14220ctaatttgtt gcgtatagca gcatctttag tactcgctcg taaacacact aattgttgta 14280cttggtctga acgcgtttat aggttgtata atgaatgcgc tcaggtttta tctgaaactg 14340tcttagctac aggtggtata tatgtgaaac ctggtggtac tagcagtgga gatgctacta 14400ctgcttatgc aaacagtgtt ttcaacataa tacaagccac atctgctaat gttgcgcgtc 14460ttttgagtgt tataacgcgt gatattgtat atgatgacat taagagcttg cagtatgaat 14520tgtaccagca ggtttatagg cgagtcaatt ttgacccagc atttgttgaa aagttttatt 14580cttatttgtg taagaatttc tcattgatga tcttgtctga cgacggtgtt gtttgttata 14640acaacacatt agccaaacaa ggtcttgtag cagatatttc tggttttaga gaagttctct 14700actatcagaa caatgttttt atggctgatt ctaaatgttg ggttgaacca gatttagaaa 14760aaggcccaca tgaattttgt tcacagcaca caatgttagt ggaggttgat ggtgagccta 14820gatacttgcc atatccagac ccatcacgta ttttgtgtgc atgtgttttt gtagatgatt 14880tggataagac agaatctgtg gctgttatgg agcgttatat cgctcttgcc atagatgcgt 14940acccactagt acatcatgaa aatgaggagt acaagaaggt attctttgtg cttctttcat 15000acatcagaaa actctatcaa

gagctttctc agaatatgct tatggactac tcttttgtaa 15060tggatataga taagggtagt aaattttggg aacaggagtt ctatgaaaat atgtatagag 15120cccctacaac attacagtct tgtggcgttt gtgtagtgtg taatagtcaa actatattgc 15180gctgtggtaa ttgtattcgc aaaccatttt tgtgttgtaa gtgttgctat gaccatgtca 15240tgcacacaga ccacaaaaat gttttgtcta taaatcctta catttgctca cagccaggtt 15300gtggtgaagc agatgttact aaattgtacc tcggaggtat gtcatacttc tgcggtaatc 15360ataaaccaaa gttatcaata ccgttagtat ctaatggtac agtgtttgga atttacaggg 15420ctaattgtgc aggtagcgaa aatgttgatg attttaatca actagctact actaattggt 15480ctactgtgga accttatatt ttggcaaatc gttgtgtaga ttcgttgaga cgctttgctg 15540cagagacagt aaaagctaca gaagaattac ataagcaaca atttgctagt gcagaagtga 15600gagaagtact ctcagatcgt gaattgattc tgtcttggga gccaggtaaa accaggcctc 15660cattgaatag aaattatgtt ttcactggct ttcactttac tagaactagt aaagttcagc 15720tcggtgattt tacatttgaa aaaggtgaag gtaaggacgt tgtctattat cgagcgacgt 15780ctactgctaa attgtctgtt ggagacattt ttgttttaac ctcacacaat gttgtttctc 15840ttatagcgcc aacgttgtgt cctcagcaaa ccttttctag gtttgtgaat ttaagaccta 15900atgtgatggt acctgcgtgt tttgtaaata acattccatt gtaccattta gtaggcaagc 15960agaagcgtac tacagtacaa ggccctcctg gcagtggtaa atcccatttt gctataggat 16020tggcggctta ctttagtaac gcccgtgtcg tttttactgc atgctctcat gcagctgttg 16080atgctttatg tgaaaaagct tttaagtttc ttaaagtaga tgattgcact cgtatagtac 16140ctcaaaggac tactatcgat tgcttctcta agtttaaagc taatgacaca ggcaaaaagt 16200acatttttag tactattaat gccttgccag aagttagttg tgacattctt ttggttgacg 16260aggttagtat gttgaccaat tacgaattgt cttttattaa tggtaagata aactatcaat 16320atgttgtgta tgtaggtgat cctgctcaat taccggcgcc tcgtacgttg cttaacggtt 16380cactctctcc aaaggattat aatgttgtca caaaccttat ggtttgtgtt aaacctgaca 16440ttttccttgc aaagtgttac cgttgtccta aagaaattgt agatactgtt tctactcttg 16500tatatgatgg aaagtttatt gcaaataacc cggaatcacg tcagtgtttc aaggttatag 16560ttaataatgg taattctgat gtaggacatg aaagtggctc agcctacaac ataactcaat 16620tagaatttgt gaaagatttt gtctgtcgca ataaggaatg gcgggaagca acattcattt 16680caccttataa tgctatgaac cagagagcct accgtatgct tggacttaat gttcagacag 16740tagactcgtc tcaaggttcg gagtatgatt atgttatctt ttgtgttact gcagattcgc 16800agcatgcact gaatattaac agattcaatg tagcgcttac aagagccaag cgtggtatac 16860tagttgtcat gcgtcagcgt gatgaactat attcagctct taagtttata gagcttgata 16920gtgtagcaag tctgcaaggt acaggcttgt ttaaaatttg caacaaagag tttagtggtg 16980ttcacccagc ttatgcagtc acaactaagg ctcttgctgc aacttataaa gttaatgatg 17040aacttgctgc acttgttaac gtggaagctg gttcagaaat aacatataaa catcttattt 17100ctttgttagg gtttaagatg agtgttaatg ttgaaggctg ccacaacatg tttataacac 17160gtgatgaggc tatccgcaac gtaagaggtt gggtaggttt tgatgtagaa gcaacacatg 17220cttgcggtac taacattggt actaacctgc ctttccaagt aggtttctct actggtgcag 17280actttgtagt tacgcctgag ggacttgtag atacttcaat aggcaataat tttgagcctg 17340tgaattctaa agcacctcca ggtgaacaat ttaatcactt gagagcgtta ttcaaaagtg 17400ctaaaccttg gcatgttgta aggccaagga ttgtgcaaat gttagcggat aacctgtgca 17460acgtttcaga ttgtgtagtg tttgtcacgt ggtgtcatgg cctagaacta accactttgc 17520gctattttgt taaaataggc aaggaccaag tttgttcttg cggttctaga gcaacaactt 17580ttaattctca tactcaggct tatgcttgtt ggaagcattg cttgggtttt gattttgttt 17640ataatccact cttagtggat attcaacagt ggggttattc tggtaaccta caatttaacc 17700atgatttgca ttgtaatgtg catggacacg cacatgtagc ttctgcggat gctattatga 17760cgcgttgtct tgcaattaat aatgcatttt gtcaagatgt caactgggat ttaacttacc 17820ctcatatagc aaatgaggat gaagtcaatt ctagctgtag atatttacaa cgcatgtatc 17880ttaatgcatg tgttgatgct cttaaagtta acgttgtcta tgatataggc aaccctaaag 17940gtataaaatg tgttagacgt ggagacttaa attttagatt ctatgataag aatccaatag 18000tacccaatgt caagcagttt gagtatgact ataatcagca caaagataag tttgctgatg 18060gtctttgtat gttttggaat tgtaatgtgg attgttatcc cgacaattcc ttagtttgta 18120ggtacgacac acgaaatttg agtgtgttta acctacctgg ttgtaatggt ggtagcttgt 18180atgttaacaa gcatgcattc cacacaccta aatttgatcg cactagcttt cgtaatttga 18240aagctatgcc attctttttc tatgactcat cgccttgcga gaccattcaa ttggatggag 18300ttgcgcaaga ccttgtgtca ttagctacga aagattgtat cacaaaatgc aacataggcg 18360gtgctgtttg taaaaagcac gcacaaatgt atgcagattt tgtgacttct tataatgcag 18420ctgttactgc tggttttact ttttgggtta ctaataattt taacccatat aatttgtgga 18480aaagtttttc agctctccag tctatcgaca atattgctta taatatgtat aagggtggtc 18540attatgatgc tattgcagga gaaatgccca ctatcgtaac tggagataaa gtttttgtta 18600tagatcaagg cgtagaaaaa gcagtttttt ttaatcaaac aattctgcct acatctgtag 18660cgtttgagct gtatgcgaag agaaatattc gcacactgcc aaacaaccgt attttgaaag 18720gtttgggtgt agatgtgact aatggatttg taatttggga ttacacgaac caaacaccac 18780tataccgtaa tactgttaag gtatgtgcat atacagacat agaaccaaat ggcctaatag 18840tgctgtatga tgatagatat ggtgattacc agtcttttct agctgctgat aatgctgttt 18900tagtttctac acagtgttac aagcggtatt cgtatgtaga aataccgtca aacctgcttg 18960ttcagaacgg tattccgtta aaagatggag cgaacctgta tgtttataag cgtgttaatg 19020gtgcgtttgt tacgctacct aacacattaa acacacaggg tcgcagttat gaaacttttg 19080aacctcgtag tgatgttgag cgtgattttc tcgacatgtc tgaggagagt tttgtagaaa 19140agtatggtaa agaattaggt ctacagcaca tactgtatgg tgaagttgat aagccccaat 19200taggtggttt acacactgtt ataggtatgt gcagactttt acgtgcgaat aagttgaacg 19260caaagtctgt tactaattct gattctgatg tcatgcaaaa ttattttgta ttggcagaca 19320atggttccta caagcaagtg tgtactgttg tggatttgct gcttgatgat ttcttagaac 19380ttcttaggaa catactgaaa gagtatggta ctaataagtc taaagttgta acagtgtcaa 19440ttgattacca tagcataaat tttatgactt ggtttgaaga tggcattatt aaaacatgtt 19500atccacagct tcaatcagca tggacgtgtg gttataatat gcctgaactt tataaagttc 19560agaattgtgt tatggaacct tgcaacattc ctaattatgg tgttggaata gcgttgccaa 19620gtggtattat gatgaatgtg gcaaagtata cacaactctg tcaatacctt tcgaaaacaa 19680caatgtgtgt accgcataat atgcgagtaa tgcattttgg agctggaagt gacaaaggag 19740tggctccagg tagtactgtt cttaaacaat ggctcccaga agggacactc cttgtcgata 19800atgatattgt agactatgtg tctgatgcac atgtttctgt gctttcagat tgcaataaat 19860ataagacaga gcacaagttt gatcttgtga tatctgatat gtatacagac aatgattcaa 19920aaagaaagca tgaaggcgtg atagccaata atggcaatga tgacgttttc atatatctct 19980caagttttct tcgtaataat ttggctctag gtggtagttt tgctgtaaaa gtgacagaga 20040caagttggca cgaagtttta tatgacattg cacaggattg tgcatggtgg acaatgtttt 20100gtacagcagt gaatgcctct tcttcagaag cattcttggt tggtgttaat tatttgggtg 20160caagtgaaaa ggttaaggtt agtggaaaaa cgctgcacgc aaattatata ttttggagga 20220attgtaatta tttacaaacc tctgcttata gtatatttga cgttgctaag tttgatttga 20280gattgaaagc aacaccagtt gttaatttga aaactgaaca aaagacagac ttagtcttta 20340atttaattaa gtgtggtaag ttactggtaa gagatgttgg taacacctct tttactagtg 20400actcttttgt gtgtactatg tagtgctgct ttgtatgaca gtagttctta cgtttactac 20460taccaaagtg cctttagacc acctaatggt tggcatttac acgggggtgc ttatgcggta 20520gttaatattt ctagcgaatc taataatgca ggctcttcac ctgggtgtat tgttggtact 20580attcatggtg gtcgtgttgt taatgcttct tctatagcta tgacggcacc gtcatcaggt 20640atggcttggt ctagcagtca gttttgtact gcacactgta acttttcaga tactacagtg 20700tttgttacac attgttataa atatgatggg tgtcctataa ctggcatgct tcaaaagaat 20760tttttacgtg tttctgctat gaaaaatggc cagcttttct ataatttaac agttagtgta 20820gctaagtacc ctacttttaa atcatttcag tgtgttaata atttaacatc cgtatattta 20880aatggtgatc ttgtttacac ctctaatgag accacagatg ttacatctgc aggtgtttat 20940tttaaagctg gtggacctat aacttataaa gttatgagag aagttaaagc cctggcttat 21000tttgttaatg gtactgcaca agatgttatt ttgtgtgatg gatcacctag aggcttgtta 21060gcatgccagt ataatactgg caatttttca gatggctttt atccttttat taatagtagt 21120ttagttaagc agaagtttat tgtctatcgt gaaaatagtg ttaatactac ttttacgtta 21180cacaatttca cttttcataa tgagactggc gccaacccta atcctagtgg tgttcagaat 21240attcaaactt accaaacaca aacagctcag agtggttatt ataattttaa tttttccttt 21300ctgagtagtt ttgtttataa ggagtctaat tttatgtatg gatcttatca cccaagttgt 21360aattttagac tagaaactat taataatggc ttgtggttta attcactttc agtttcaatt 21420gcttacggtc ctcttcaagg tggttgcaag caatctgtct ttagtggtag agcaacttgt 21480tgttatgctt attcatatgg aggtccttcg ctgtgtaaag gtgtttattc aggtgagtta 21540gatcttaatt ttgaatgtgg actgttagtt tatgttacta agagcggtgg ctctcgtata 21600caaacagcca ctgaaccgcc agttataact cgacacaatt ataataatat tactttaaat 21660acttgtgttg attataatat atatggcaga actggccaag gttttattac taatgtaacc 21720gactcagctg ttagttataa ttatctagca gacgcaggtt tggctatttt agatacatct 21780ggttccatag acatctttgt tgtacaaggt gaatatggtc ttacttatta taaggttaac 21840ccttgcgaag atgtcaacca gcagtttgta gtttctggtg gtaaattagt aggtattctt 21900acttcacgta atgagactgg ttctcagctt cttgagaacc agttttacat taaaatcact 21960aatggaacac gtcgttttag acgttctatt actgaaaatg ttgcaaattg cccttatgtt 22020agttatggta agttttgtat aaaacctgat ggttcaattg ccacaatagt accaaaacaa 22080ttggaacagt ttgtggcacc tttacttaat gttactgaaa atgtgctcat acctaacagt 22140tttaatttaa ctgttacaga tgagtacata caaacgcgta tggataaggt ccaaattaat 22200tgtctgcagt atgtttgtgg caattctctg gattgtagag atttgtttca acaatatggg 22260cctgtttgtg acaacatatt gtctgtagta aatagtattg gtcaaaaaga agatatggaa 22320cttttgaatt tctattcttc tactaaaccg gctggtttta atacaccatt tcttagtaat 22380gttagcactg gtgagtttaa tatttctctt ctgttaacaa ctcctagtag tcctagaagg 22440cgttctttta ttgaagacct tctatttaca agcgttgaat ctgttggatt accaacagat 22500gacgcataca aaaattgcac tgcaggacct ttaggttttc ttaaggacct tgcgtgtgct 22560cgtgaatata atggtttgct tgtgttgcct cccattataa cagcagaaat gcaaattttg 22620tatactagtt ctctagtagc ttctatggct tttggtggta ttactgcagc tggtgctata 22680ccttttgcca cacaactgca ggctagaatt aatcacttgg gtattaccca gtcacttttg 22740ttgaagaatc aagaaaaaat tgctgcttcc tttaataagg ccattggtcg tatgcaggaa 22800ggttttagaa gtacatctct agcattacaa caaattcaag atgttgttaa taagcagagt 22860gctattctta ctgagactat ggcatcactt aataaaaatt ttggtgctat ttcttctatg 22920attcaagaaa tctaccagca acttgacgcc atacaagcaa atgctcaagt ggatcgtctt 22980ataactggta gattgtcatc actttctgtt ttagcatctg ctaagcaggc ggagcatatt 23040agagtgtcac aacagcgtga gttagctact cagaaaatta atgagtgtgt taagtcacag 23100tctattaggt actccttttg tggtaatgga cgacatgttc taaccatacc gcaaaatgca 23160cctaatggta tagtgtttat acacttttct tatactccag atagttttgt taatgttact 23220gcaatagtgg gtttttgtgt aaagccagct aatgctagtc agtatgcaat agtacccgct 23280aatggtaggg gtatttttat acaagttaat ggtagttact acatcacagc acgagatatg 23340tatatgccaa gagctattac tgcaggagat atagttacgc ttacttcttg tcaagcaaat 23400tatgtaagtg taaataagac cgtcattact acattcgtag acaatgatga ttttgatttt 23460aatgacgaat tgtcaaaatg gtggaatgac actaagcatg agctaccaga ctttgacaaa 23520ttcaattaca cagtacctat acttgacatt gatagtgaaa ttgatcgtat tcaaggcgtt 23580atacagggtc ttaatgactc tttaatagac cttgaaaaac tttcaatact caaaacttat 23640attaagtggc cttggtatgt gtggttagcc atagcttttg ccactattat cttcatctta 23700atactaggat gggttttctt catgactgga tgttgtggtt gttgttgtgg atgctttggc 23760attatgcctc taatgagtaa gtgtggtaag aaatcttctt attacacgac ttttgataac 23820gatgtggtaa cttaacaata cagacctaaa aagtctgttt aatgattcaa agtcccacgt 23880ccttcctaat agtattaatt tttctttggt gtaaacttgt actaagttgt tttagagagt 23940ttattatagc gctccaacaa ctaatacaag ttttactcca aattatcaat agtaacttac 24000agcctagact gaccctttgt cacagtctag actaatgtta aacttagaag caattattga 24060aactggtgag caagtgattc aaaaaatcag tttcaattta cagcatattt caagtgtatt 24120aaacacagaa gtatttgacc cctttgacta ttgttattac agaggaggta atttttggga 24180aatagagtca gctgaagatt gttcaggtga tgatgaattt attgaataag tcgctagagg 24240aaaatggaag ttttctaaca gcgctttata tatttgtagg atttttagca ctttatcttc 24300taggtagagc acttcaagca tttgtacagg ctgctgatgc ttgttgttta ttttggtata 24360catgggtagt aattccagga gctaagggta cagcctttgt atataagtat acatatggta 24420gaaaacttaa caatccggaa ttagaagcag ttattgtcaa cgagtttcct aagaacggtt 24480ggaataataa aaatccagca aattttcaag atgtccaacg agacaaattg tactcttgac 24540tttgaacagt cagttgagct ttttaaagag tataatttat ttataactgc attcttgttg 24600ttcttaacca taatacttca gtatggctat gcaacaagaa gtaagtttat ttatatactg 24660aaaatgatag tgttatggtg cttttggccc cttaacattg cagtaggtgt aatttcatgt 24720atatacccac caaacacagg aggtcttgtc gcagcgataa tacttacagt gtttgcgtgt 24780ctgtcttttg taggttattg gatccagagt attagactct ttaagcggtg taggtcatgg 24840tggtcattta acccagaatc taatgccgta ggttcaatac tcctaactaa tggtcaacaa 24900tgtaattttg ctatagagag tgtgccaatg gtgctttctc caattataaa gaatggtgtt 24960ctttattgtg agggtcagtg gcttgctaag tgtgaaccag accacttgcc taaagatata 25020tttgtttgta caccggatag acgtaatatc taccgtatgg tgcagaaata tactggtgac 25080caaagcggaa ataagaaacg gtttgctacg tttgtctatg caaagcagtc agtagatact 25140ggcgagctag aaagtgtagc aacaggaggg agtagtcttt acacctaaat gtgtgtgtgt 25200agagagtatt taaaattatt ctttaatagt gcctctattt taagagcgca taatagtatt 25260atttttgagg atattaatat aaatcctctc tgttttatac tctcttttca agagctatta 25320tttaaaaaac agtttttcca ctcttttgtg ccaaaaacta ttgttgttaa tggtgtaacc 25380tttcaagtag ataatggaaa agtctactac gaaggaaaac caatttttca gaaaggttgt 25440tgtaggttgt ggttgagtta taaaaaagat taaactacct actacactta tttttataag 25500aggcgtttta tcttacaagc gcttaataaa tacggacgat gaaatggctg actagttttg 25560taagggcagt tatttcatgt tataaacccc tattattaac tcaattaaga gtattagata 25620ggttaatctt agatcatgga ccaaaacaca tcttaacgtg tgttaggtgc gtgattttgt 25680ttcaattaga tttagtttat aggttggcgt atacgcctac tcaatcgctg gtatgaataa 25740tagtaaagat aatccttttt gcggagcaat agcaagaaaa gcgcgaattt atctgagaga 25800aggattagat tgtgtttact ttcttaacaa agcaggacaa gcagagtctt gtcccgcgtg 25860tacctctcta gtattccagg ggaaaacttg tgaggaacac aaatataata ataatctttt 25920gtcatggcaa gcggtaaggc aactggaaag acagatgccc cagctccagt catcaaacta 25980ggaggaccaa agccacctaa agttggttct tctggaaatg tatcttggtt tcaagcaata 26040aaagccaaga agttaaattc acctccgcct aagtttgaag gtagcggtgt tcctgataat 26100gaaaatctaa aaccaagtca gcagcatgga tattggagac gccaagctag gtttaagcca 26160ggtaaaggtg gaagaaaacc agtcccagat gcttggtatt tttactatac tggaacagga 26220ccagccgcta acctgaattg gggtgatagc caagatggta tagtgtgggt tgctggtaag 26280ggtgctgata ctaaatttag atctaatcag ggtactcgtg actctgacaa gtttgaccaa 26340tatccgctac ggttttcaga cggaggacct gatggtaatt tccgttggga tttcattcct 26400ctgaatcgtg gcaggagtgg gagatcaaca gcagcttcat cagcagcatc tagtagagca 26460ccatcacgtg aagtttcgcg tggtcgcagg agtggttctg aagatgatct tattgctcgt 26520gcagcaagga taattcagga tcagcagaag aagggttctc gcattacaaa ggctaaggct 26580gatgaaatgg ctcaccgccg gtattgcaag cgcactattc cacctaatta taaggttgat 26640caagtgtttg gtccccgtac taaaggtaag gagggaaatt ttggtgatga caagatgaat 26700gaggaaggta ttaaggatgg gcgcgttaca gcaatgctca acctagttcc tagcagccat 26760gcttgtcttt tcggaagtag agtgacgccc agacttcaac cagatgggct gcacttgaaa 26820tttgaattta ctactgtggt cccacgtgat gatccgcagt ttgataatta tgtaaaaatt 26880tgtgatcagt gtgttgatgg tgtaggaaca cgtccaaaag atgatgaacc aagaccaaag 26940tcacgctcaa gttcaagacc tgcaacaaga ggaaattctc cagcgccaag acagcagcgc 27000cctaagaagg agaaaaagcc aaagaagcag gatgatgaag tggataaagc attgacctca 27060gatgaggaga ggaacaatgc acagctggaa tttgatgatg aacccaaggt aattaactgg 27120ggggattcag ccctaggaga gaatgaactt tgagtaaaat tcaatagtaa gagttaagga 27180agataggcat gtagcttgat tacctacatg tctatcgcca gggaaatgtc taatttgtct 27240acttagtagc ctggaaacga acggtagacc cttagatttt aatttagttt aatttttagt 27300ttagtttaag ttagtttaga gtaggtataa agatgccagt gccggggcca cgcggagtac 27360gaccgagggt acagcactag gacgcccatt aggggaagag ctaaatttta gtttaagtta 27420agtttaattg gctatgtata gttaaaattt ataggctagt atagagttag agcaaaaaaa 27480aaaaaaaaaa aaaaaaaaaa 275002709DNAAvian infectious bronchitis virus 2cttaacaata cagacctaaa aagtctgttt aatgattcaa agtcccacgt ccttcctaat 60agtattaatt tttctttggt gtaaacttgt actaagttgt tttagagagt ttattatagc 120gctccaacaa ctaatacaag ttttactcca aattatcaat agtaacttac agcctagact 180gaccctttgt cacagtctag actaatgtta aacttagaag caattattga aactggtgag 240caagtgattc aaaaaatcag tttcaattta cagcatattt caagtgtatt aaacacagaa 300gtatttgacc cctttgacta ttgttattac agaggaggta atttttggga aatagagtca 360gctgaagatt gttcaggtga tgatgaattt attgaataag tcgctagagg aaaatggaag 420ttttctaaca gcgctttata tatttgtagg atttttagca ctttatcttc taggtagagc 480acttcaagca tttgtacagg ctgctgatgc ttgttgttta ttttggtata catgggtagt 540aattccagga gctaagggta cagcctttgt atataagtat acatatggta gaaaacttaa 600caatccggaa ttagaagcag ttattgtcaa cgagtttcct aagaacggtt ggaataataa 660aaatccagca aattttcaag atgtccaacg agacaaattg tactcttga 709357PRTAvian infectious bronchitis virus 3Met Ile Gln Ser Pro Thr Ser Phe Leu Ile Val Leu Ile Phe Leu Trp 1 5 10 15 Cys Lys Leu Val Leu Ser Cys Phe Arg Glu Phe Ile Ile Ala Leu Gln 20 25 30 Gln Leu Ile Gln Val Leu Leu Gln Ile Ile Asn Ser Asn Leu Gln Pro 35 40 45 Arg Leu Thr Leu Cys His Ser Leu Asp 50 55 464PRTAvian infectious bronchitis virus 4Met Leu Asn Leu Glu Ala Ile Ile Glu Thr Gly Glu Gln Val Ile Gln 1 5 10 15 Lys Ile Ser Phe Asn Leu Gln His Ile Ser Ser Val Leu Asn Thr Glu 20 25 30 Val Phe Asp Pro Phe Asp Tyr Cys Tyr Tyr Arg Gly Gly Asn Phe Trp 35 40 45 Glu Ile Glu Ser Ala Glu Asp Cys Ser Gly Asp Asp Glu Phe Ile Glu 50 55 60 54782DNAAvian infectious bronchitis virus 5ggtaaaactg tcacctttgg agaaactact gtgcaagaaa taccaccacc tgatgttgtg 60tttattaagg ttagcattga gtgttgtggt gaaccatgga atacaatctt caaaaaggct 120tataaggagc ccattgaagt agagacagac ctcacagttg aacaattgct ctctgtggtc 180tatgagaaaa tgtgtgatga tctcaagctg tttccggagg ctccagaacc accaccattt 240gagaatgtca cacttgttga taagaatggt aaagatttgg attgcataaa atcatgccat 300ctgatctatc gtgattatga gagcgatgat gacatcgagg aagaagatgc agaagaatgt 360gacacggatt caggtgatgc tgaggagtgt gacactaatt cagaatgtga agaagaagat 420gaggatacta aagtgttggc tcttatacaa gacccggcaa gtaacaaata tcctctgcct 480cttgatgatg attatagcgt ctacaatgga tgtattgttc ataaggacgc tctcgatgtt 540gtgaatttac catctggtga agaaaccttt gttgtcaata actgctttga aggggctgtt 600aaagctcttc cgcagaaagt tattgatgtt ctaggtgact ggggtgaggc tgttgatgcg 660caagaacaat tgtgtcaaca agaatcaact cgggtcatat ctgagaaatc agttgagggt 720tttactggta gttgtgatgc aatggctgaa caagctattg ttgaagagca ggaaatagta 780cctgttgttg aacaaagtca ggatgtagtt gtttttacac ctgcagacct agaagttgtt 840aaagaaacag cagaagaggt tgatgagttt attctcattt ctgctgtccc

taaagaagaa 900gttgtgtctc aggagaaaga ggagccacag gttgagcaag agcctaccct agttgttaaa 960gcacaacgtg agaagaaggc taaaaagttc aaagttaaac cagctacatg tgaaaaaccc 1020aaatttttgg agtacaaaac atgtgtgggt gatttggctg ttgtaattgc caaagcattg 1080gatgagttta aagagttctg cattgtaaac gctgcaaatg agcacatgtc gcatggtggt 1140ggcgttgcaa aggcaattgc agacttttgt ggaccggact ttgttgaata ttgcgcggac 1200tatgttaaga aacatggtcc acagcaaaaa cttgtcacac cttcatttgt taaaggcatt 1260caatgtgtga ataatgttgt aggacctcgc catggagaca gcaacttgcg tgagaagctt 1320gttgctgctt acaagagtgt tcttgtaggt ggagtggtta actatgttgt gccagttctc 1380tcatcaggga tttttggtgt agattttaaa atatcaatag atgctatgcg cgaagctttt 1440aaaggttgtg ccatacgcgt tcttttattt tctctgagtc aagaacacat cgattatttc 1500gatgcaactt gtaagcagaa gacaatttat cttacggagg atggtgttaa ataccgctct 1560gttgttttaa aacctggtga ttctttgggt caatttggac aggtttttgc aagaaataag 1620gtagtctttt cggctgatga tgttgaggat aaagaaatcc tctttatacc cacaactgac 1680aagactattc ttgaatatta tggtttagat gcgcaaaagt atgtaacata tttgcaaacg 1740cttgcgcaga aatgggatgt tcaatataga gacaattttg ttatattaga gtggcgtgac 1800ggaaattgct ggattagttc agcaatagtt ctccttcaag ctgctaaaat tagatttaaa 1860ggttttcttg cagaagcatg ggctaaactg ttgggtggag atcctacaga ctttgttgcc 1920tggtgttatg caagttgcaa tgctaaagta ggtgattttt cagatgctaa ttggcttttg 1980gccaatttag cagaacattt tgacgcagat tacacaaatg cacttcttaa gaagtgtgtg 2040tcgtgcaatt gtggtgttaa gagttatgaa cttaggggtc ttgaagcctg tattcagcca 2100gttcgagcac ctaatcttct acattttaaa acgcaatatt caaattgccc aacctgtggt 2160gcaagtagta cggatgaagt aatagaagct tcattaccgt acttattgct ttttgctact 2220gatggtcctg ctacagttga ttgtgatgaa aatgctgtag ggactgttgt tttcattggc 2280tctactaata gtggccattg ttatacacaa gccgatggta aggcttttga caatcttgct 2340aaggatagaa aatttggaag gaagtcgcct tacattacag caatgtatac acgtttttct 2400cttaggagtg aaaatcccct acttgttgtt gaacatagta agggtaaagc taaagtagta 2460aaagaagatg tttctaacct tgctactagt tctaaagcca gttttgacga tcttactgac 2520tttgaacagt ggtatgatag caacatctat gagagtctta aagtgcagga gacacctgat 2580aatcttgatg aatatgtgtc atttacgaca aaggaagatt ctaagttgcc actgacactt 2640aaagttagag gtatcaaatc agttgttgac tttaggtcta aggatggttt tacttataag 2700ttaacacctg atactgatga aaattcaaaa acaccagtct actacccagt cttggattct 2760attagtctta gggcaatatg ggttgaaggc agtgctaatt ttgttgttgg gcatccaaat 2820tattatagta agtctctccg aattcccacg ttttgggaaa atgccgagag ctttgttaaa 2880atgggttata aaattgatgg tgtaactatg ggcctttggc gtgcagaaca ccttaataaa 2940cctaatttgg agagaatttt taacattgct aagaaagcta ttgttggatc tagtgttgtt 3000actacgcagt gtggtaaaat actagttaaa gcagctacat acgttgccga taaagtaggt 3060gatggtgtag ttcgcaatat tacagataga attaagggtc tttgtggatt cacacgtggc 3120cattttgaaa agaaaatgtc cctacaattt ctaaagacac ttgtgttctt tttcttttat 3180ttcttaaagg ctagtgctaa gagtttagtt tctagctata agattgtgtt atgtaaggtg 3240gtgtttgcta ccttacttat agtgtggttt atatacacaa gtaatccagt agtgtttact 3300ggaatacgtg tgctagactt cctatttgaa ggttctttat gtggtcctta taatgactac 3360ggtaaagatt cttttgatgt gttacgctat tgtgcaggtg attttacttg tcgtgtgtgt 3420ttacatgata gagattcact tcatctgtac aaacatgctt atagcgtaga acaaatttat 3480aaggatgcag cttctggcat taactttaat tggaattggc tttatttggt ctttctaata 3540ttatttgtta agccagtggc aggttttgtt attatttgtt attgtgttaa gtatttggta 3600ttgagttcaa ctgtgttgca aactggtgta ggttttctag attggtttgt aaaaacagtt 3660tttacccatt ttaattttat gggagcggga ttttatttct ggctctttta caagatatac 3720gtacaagtgc atcatatatt gtactgtaag gatgtaacat gtgaagtgtg caagagagtt 3780gcacgcagca acaggcaaga ggttagcgtt gtagttggtg gacgcaagca aatagtgcat 3840gtttacacta attctggcta taacttttgt aagagacata attggtattg tagaaattgt 3900gatgattatg gtcaccaaaa tacatttatg tcccctgaag ttgctggcga gctttctgaa 3960aagcttaagc gccatgttaa acctacagca tatgcttacc acgttgtgta tgaggcatgc 4020gtggttgatg attttgttaa tttaaaatat aaggctgcaa ttcctggtaa ggataatgca 4080tcttctgctg ttaagtgttt cagtgttaca gattttttaa agaaagctgt ttttcttaag 4140gaggcattga aatgtgaaca aatatctaat gatggtttta tagtgtgtaa tacacagagt 4200gcgcatgcac tagaggaagc aaagaatgca gccgtctatt atgcgcaata tctgtgtaag 4260ccaatactta tacttgacca ggcactttat gagcaattaa tagtagagcc tgtgtctaag 4320agtgttatag ataaagtgtg tagcattttg tctaatataa tatctgtaga tactgcagct 4380ttaaattata aggcaggcac acttcgtgat gctctgcttt ctattactaa agacgaagaa 4440gccgtagata tggctatctt ctgccacaat catgaagtgg aatacactgg tgacggtttt 4500actaatgtga taccgtcata tggtatggac actgataagt tgacacctcg tgatagaggg 4560tttttgataa atgcagatgc ttctattgct aatttaagag tcaaaaatgc tcctccggta 4620gtatggaagt tttctgatct tattaaattg tctgacagtt gccttaaata tttaatttca 4680gctactgtca agtcaggagg tcgtttcttt ataacaaagt ctggtgctaa acaagttatt 4740tcttgtcata cccagaaact gttggtagag aaaaaggcag gt 478261594PRTAvian infectious bronchitis virus 6Gly Lys Thr Val Thr Phe Gly Glu Thr Thr Val Gln Glu Ile Pro Pro 1 5 10 15 Pro Asp Val Val Phe Ile Lys Val Ser Ile Glu Cys Cys Gly Glu Pro 20 25 30 Trp Asn Thr Ile Phe Lys Lys Ala Tyr Lys Glu Pro Ile Glu Val Glu 35 40 45 Thr Asp Leu Thr Val Glu Gln Leu Leu Ser Val Val Tyr Glu Lys Met 50 55 60 Cys Asp Asp Leu Lys Leu Phe Pro Glu Ala Pro Glu Pro Pro Pro Phe 65 70 75 80 Glu Asn Val Thr Leu Val Asp Lys Asn Gly Lys Asp Leu Asp Cys Ile 85 90 95 Lys Ser Cys His Leu Ile Tyr Arg Asp Tyr Glu Ser Asp Asp Asp Ile 100 105 110 Glu Glu Glu Asp Ala Glu Glu Cys Asp Thr Asp Ser Gly Asp Ala Glu 115 120 125 Glu Cys Asp Thr Asn Ser Glu Cys Glu Glu Glu Asp Glu Asp Thr Lys 130 135 140 Val Leu Ala Leu Ile Gln Asp Pro Ala Ser Asn Lys Tyr Pro Leu Pro 145 150 155 160 Leu Asp Asp Asp Tyr Ser Val Tyr Asn Gly Cys Ile Val His Lys Asp 165 170 175 Ala Leu Asp Val Val Asn Leu Pro Ser Gly Glu Glu Thr Phe Val Val 180 185 190 Asn Asn Cys Phe Glu Gly Ala Val Lys Ala Leu Pro Gln Lys Val Ile 195 200 205 Asp Val Leu Gly Asp Trp Gly Glu Ala Val Asp Ala Gln Glu Gln Leu 210 215 220 Cys Gln Gln Glu Ser Thr Arg Val Ile Ser Glu Lys Ser Val Glu Gly 225 230 235 240 Phe Thr Gly Ser Cys Asp Ala Met Ala Glu Gln Ala Ile Val Glu Glu 245 250 255 Gln Glu Ile Val Pro Val Val Glu Gln Ser Gln Asp Val Val Val Phe 260 265 270 Thr Pro Ala Asp Leu Glu Val Val Lys Glu Thr Ala Glu Glu Val Asp 275 280 285 Glu Phe Ile Leu Ile Ser Ala Val Pro Lys Glu Glu Val Val Ser Gln 290 295 300 Glu Lys Glu Glu Pro Gln Val Glu Gln Glu Pro Thr Leu Val Val Lys 305 310 315 320 Ala Gln Arg Glu Lys Lys Ala Lys Lys Phe Lys Val Lys Pro Ala Thr 325 330 335 Cys Glu Lys Pro Lys Phe Leu Glu Tyr Lys Thr Cys Val Gly Asp Leu 340 345 350 Ala Val Val Ile Ala Lys Ala Leu Asp Glu Phe Lys Glu Phe Cys Ile 355 360 365 Val Asn Ala Ala Asn Glu His Met Ser His Gly Gly Gly Val Ala Lys 370 375 380 Ala Ile Ala Asp Phe Cys Gly Pro Asp Phe Val Glu Tyr Cys Ala Asp 385 390 395 400 Tyr Val Lys Lys His Gly Pro Gln Gln Lys Leu Val Thr Pro Ser Phe 405 410 415 Val Lys Gly Ile Gln Cys Val Asn Asn Val Val Gly Pro Arg His Gly 420 425 430 Asp Ser Asn Leu Arg Glu Lys Leu Val Ala Ala Tyr Lys Ser Val Leu 435 440 445 Val Gly Gly Val Val Asn Tyr Val Val Pro Val Leu Ser Ser Gly Ile 450 455 460 Phe Gly Val Asp Phe Lys Ile Ser Ile Asp Ala Met Arg Glu Ala Phe 465 470 475 480 Lys Gly Cys Ala Ile Arg Val Leu Leu Phe Ser Leu Ser Gln Glu His 485 490 495 Ile Asp Tyr Phe Asp Ala Thr Cys Lys Gln Lys Thr Ile Tyr Leu Thr 500 505 510 Glu Asp Gly Val Lys Tyr Arg Ser Val Val Leu Lys Pro Gly Asp Ser 515 520 525 Leu Gly Gln Phe Gly Gln Val Phe Ala Arg Asn Lys Val Val Phe Ser 530 535 540 Ala Asp Asp Val Glu Asp Lys Glu Ile Leu Phe Ile Pro Thr Thr Asp 545 550 555 560 Lys Thr Ile Leu Glu Tyr Tyr Gly Leu Asp Ala Gln Lys Tyr Val Thr 565 570 575 Tyr Leu Gln Thr Leu Ala Gln Lys Trp Asp Val Gln Tyr Arg Asp Asn 580 585 590 Phe Val Ile Leu Glu Trp Arg Asp Gly Asn Cys Trp Ile Ser Ser Ala 595 600 605 Ile Val Leu Leu Gln Ala Ala Lys Ile Arg Phe Lys Gly Phe Leu Ala 610 615 620 Glu Ala Trp Ala Lys Leu Leu Gly Gly Asp Pro Thr Asp Phe Val Ala 625 630 635 640 Trp Cys Tyr Ala Ser Cys Asn Ala Lys Val Gly Asp Phe Ser Asp Ala 645 650 655 Asn Trp Leu Leu Ala Asn Leu Ala Glu His Phe Asp Ala Asp Tyr Thr 660 665 670 Asn Ala Leu Leu Lys Lys Cys Val Ser Cys Asn Cys Gly Val Lys Ser 675 680 685 Tyr Glu Leu Arg Gly Leu Glu Ala Cys Ile Gln Pro Val Arg Ala Pro 690 695 700 Asn Leu Leu His Phe Lys Thr Gln Tyr Ser Asn Cys Pro Thr Cys Gly 705 710 715 720 Ala Ser Ser Thr Asp Glu Val Ile Glu Ala Ser Leu Pro Tyr Leu Leu 725 730 735 Leu Phe Ala Thr Asp Gly Pro Ala Thr Val Asp Cys Asp Glu Asn Ala 740 745 750 Val Gly Thr Val Val Phe Ile Gly Ser Thr Asn Ser Gly His Cys Tyr 755 760 765 Thr Gln Ala Asp Gly Lys Ala Phe Asp Asn Leu Ala Lys Asp Arg Lys 770 775 780 Phe Gly Arg Lys Ser Pro Tyr Ile Thr Ala Met Tyr Thr Arg Phe Ser 785 790 795 800 Leu Arg Ser Glu Asn Pro Leu Leu Val Val Glu His Ser Lys Gly Lys 805 810 815 Ala Lys Val Val Lys Glu Asp Val Ser Asn Leu Ala Thr Ser Ser Lys 820 825 830 Ala Ser Phe Asp Asp Leu Thr Asp Phe Glu Gln Trp Tyr Asp Ser Asn 835 840 845 Ile Tyr Glu Ser Leu Lys Val Gln Glu Thr Pro Asp Asn Leu Asp Glu 850 855 860 Tyr Val Ser Phe Thr Thr Lys Glu Asp Ser Lys Leu Pro Leu Thr Leu 865 870 875 880 Lys Val Arg Gly Ile Lys Ser Val Val Asp Phe Arg Ser Lys Asp Gly 885 890 895 Phe Thr Tyr Lys Leu Thr Pro Asp Thr Asp Glu Asn Ser Lys Thr Pro 900 905 910 Val Tyr Tyr Pro Val Leu Asp Ser Ile Ser Leu Arg Ala Ile Trp Val 915 920 925 Glu Gly Ser Ala Asn Phe Val Val Gly His Pro Asn Tyr Tyr Ser Lys 930 935 940 Ser Leu Arg Ile Pro Thr Phe Trp Glu Asn Ala Glu Ser Phe Val Lys 945 950 955 960 Met Gly Tyr Lys Ile Asp Gly Val Thr Met Gly Leu Trp Arg Ala Glu 965 970 975 His Leu Asn Lys Pro Asn Leu Glu Arg Ile Phe Asn Ile Ala Lys Lys 980 985 990 Ala Ile Val Gly Ser Ser Val Val Thr Thr Gln Cys Gly Lys Ile Leu 995 1000 1005 Val Lys Ala Ala Thr Tyr Val Ala Asp Lys Val Gly Asp Gly Val 1010 1015 1020 Val Arg Asn Ile Thr Asp Arg Ile Lys Gly Leu Cys Gly Phe Thr 1025 1030 1035 Arg Gly His Phe Glu Lys Lys Met Ser Leu Gln Phe Leu Lys Thr 1040 1045 1050 Leu Val Phe Phe Phe Phe Tyr Phe Leu Lys Ala Ser Ala Lys Ser 1055 1060 1065 Leu Val Ser Ser Tyr Lys Ile Val Leu Cys Lys Val Val Phe Ala 1070 1075 1080 Thr Leu Leu Ile Val Trp Phe Ile Tyr Thr Ser Asn Pro Val Val 1085 1090 1095 Phe Thr Gly Ile Arg Val Leu Asp Phe Leu Phe Glu Gly Ser Leu 1100 1105 1110 Cys Gly Pro Tyr Asn Asp Tyr Gly Lys Asp Ser Phe Asp Val Leu 1115 1120 1125 Arg Tyr Cys Ala Gly Asp Phe Thr Cys Arg Val Cys Leu His Asp 1130 1135 1140 Arg Asp Ser Leu His Leu Tyr Lys His Ala Tyr Ser Val Glu Gln 1145 1150 1155 Ile Tyr Lys Asp Ala Ala Ser Gly Ile Asn Phe Asn Trp Asn Trp 1160 1165 1170 Leu Tyr Leu Val Phe Leu Ile Leu Phe Val Lys Pro Val Ala Gly 1175 1180 1185 Phe Val Ile Ile Cys Tyr Cys Val Lys Tyr Leu Val Leu Ser Ser 1190 1195 1200 Thr Val Leu Gln Thr Gly Val Gly Phe Leu Asp Trp Phe Val Lys 1205 1210 1215 Thr Val Phe Thr His Phe Asn Phe Met Gly Ala Gly Phe Tyr Phe 1220 1225 1230 Trp Leu Phe Tyr Lys Ile Tyr Val Gln Val His His Ile Leu Tyr 1235 1240 1245 Cys Lys Asp Val Thr Cys Glu Val Cys Lys Arg Val Ala Arg Ser 1250 1255 1260 Asn Arg Gln Glu Val Ser Val Val Val Gly Gly Arg Lys Gln Ile 1265 1270 1275 Val His Val Tyr Thr Asn Ser Gly Tyr Asn Phe Cys Lys Arg His 1280 1285 1290 Asn Trp Tyr Cys Arg Asn Cys Asp Asp Tyr Gly His Gln Asn Thr 1295 1300 1305 Phe Met Ser Pro Glu Val Ala Gly Glu Leu Ser Glu Lys Leu Lys 1310 1315 1320 Arg His Val Lys Pro Thr Ala Tyr Ala Tyr His Val Val Tyr Glu 1325 1330 1335 Ala Cys Val Val Asp Asp Phe Val Asn Leu Lys Tyr Lys Ala Ala 1340 1345 1350 Ile Pro Gly Lys Asp Asn Ala Ser Ser Ala Val Lys Cys Phe Ser 1355 1360 1365 Val Thr Asp Phe Leu Lys Lys Ala Val Phe Leu Lys Glu Ala Leu 1370 1375 1380 Lys Cys Glu Gln Ile Ser Asn Asp Gly Phe Ile Val Cys Asn Thr 1385 1390 1395 Gln Ser Ala His Ala Leu Glu Glu Ala Lys Asn Ala Ala Val Tyr 1400 1405 1410 Tyr Ala Gln Tyr Leu Cys Lys Pro Ile Leu Ile Leu Asp Gln Ala 1415 1420 1425 Leu Tyr Glu Gln Leu Ile Val Glu Pro Val Ser Lys Ser Val Ile 1430 1435 1440 Asp Lys Val Cys Ser Ile Leu Ser Asn Ile Ile Ser Val Asp Thr 1445 1450 1455 Ala Ala Leu Asn Tyr Lys Ala Gly Thr Leu Arg Asp Ala Leu Leu 1460 1465 1470 Ser Ile Thr Lys Asp Glu Glu Ala Val Asp Met Ala Ile Phe Cys 1475 1480 1485 His Asn His Glu Val Glu Tyr Thr Gly Asp Gly Phe Thr Asn Val 1490 1495 1500 Ile Pro Ser Tyr Gly Met Asp Thr Asp Lys Leu Thr Pro Arg Asp 1505 1510 1515 Arg Gly Phe Leu Ile Asn Ala Asp Ala Ser Ile Ala Asn Leu Arg 1520 1525 1530 Val Lys Asn Ala Pro Pro Val Val Trp Lys Phe Ser Asp Leu Ile 1535 1540 1545 Lys Leu Ser Asp Ser Cys Leu Lys Tyr Leu Ile Ser Ala Thr Val 1550 1555 1560 Lys Ser Gly Gly Arg Phe Phe Ile Thr Lys Ser Gly Ala Lys Gln 1565 1570 1575 Val Ile Ser Cys His Thr Gln Lys Leu Leu Val Glu Lys Lys Ala 1580 1585 1590 Gly

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.