patft.uspto.gov
United States Patent | 10,130,701 |
Bickerton , et al. | November 20, 2018 |
Coronavirus
Abstract
The present invention provides a live, attenuated coronavirus comprising a variant replicase gene encoding polyproteins comprising a mutation in one or more of non-structural protein(s) (nsp)-10, nsp-14, nsp-15 or nsp-16. The coronavirus may be used as a vaccine for treating and/or preventing a disease, such as infectious bronchitis, in a subject.
Inventors: | Bickerton; Erica (Woking, GB), Keep; Sarah (Woking, GB), Britton; Paul (Woking, GB) |
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Applicant: |
Name | City | State | Country | Type |
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THE PIRBRIGHT INSTITUTE |
Pirbright, Woking |
N/A
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GB
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Assignee: | THE PIRBRIGHT INSTITUTE (Woking, Pirbright, GB) |
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Family ID: | 51494985 |
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Appl. No.: | 15/328,179 |
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Filed: | July 23, 2015 |
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PCT Filed: | July 23, 2015 |
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PCT No.: | PCT/GB2015/052124 |
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371(c)(1),(2),(4) Date: | January 23, 2017 |
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PCT Pub. No.: | WO2016/012793 |
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PCT Pub. Date: | January 28, 2016 |
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Prior Publication Data
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| Document Identifier | Publication Date |
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| US 20170216427 A1 | Aug 3, 2017 |
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Foreign Application Priority Data
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Jul 23, 2014 [GB] | | | 1413020.7 | |
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Current U.S. Class: | 1/1 |
Current CPC Class: | C12N 7/00 (20130101); C12N 9/127 (20130101); C07K 14/005 (20130101); A61K 39/215 (20130101); C12Y 207/07048 (20130101); C12N 2770/20062 (20130101); C12N 2770/20022 (20130101); C12N 2770/20034 (20130101); C12N 2770/20051 (20130101); A61K 2039/70 (20130101); C12N 2770/20021 (20130101); A61K 2039/5254 (20130101); A61K 2039/54 (20130101); C12N 2770/20071 (20130101) |
Current International Class: | A61K 39/215 (20060101); C12N 9/12 (20060101); C12N 7/00 (20060101); A61K 39/00 (20060101) |
References Cited [Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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| WO-2004/092360 | | Oct 2004 | | WO |
| WO-2005/049814 | | Jun 2005 | | WO |
| WO-2007/078203 | | Jul 2007 | | WO |
| WO-2011/004146 | | Jan 2011 | | WO |
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Other References
Sperry Journal of Virology, 2005, vol. 79, No. 6, pp. 3391-3400. cited by examiner .
Altschul et al., Basic local alignment search tool. J. Mol. Biol. 215: 403-10 (1990). cited by applicant .
Ammayapppan et al., Identification of sequence changes responsible for the attenuation of avian infectious bronchitis virus strain Arkansas DPI, Arch. Virol., 154(3):495-9 (2009). cited by applicant .
Anonymous: "EM_STD:KF377577", Oct. 30, 2013. cited by applicant .
Armesto et al., A recombinant avian infectious bronchitis virus expressing a heterologous spike gene belonging to the 4/91 serotype, PLoS One, 6(8):e24352 (2011). cited by applicant .
Armesto et al., The replicase gene of avian coronavirus infectious bronchitis virus is a determinant of pathogenicity, PLoS One, 4(10):e7384 (2009). cited by applicant .
Armesto et al., Transient dominant selection for the modification and generation of recombinant infectious bronchitis coronaviruses, Methods Mol. Biol., 454:255-73 (2008). cited by applicant .
Ausubel et al., Short Protocols in Molecular Biology, 4th edition, Chapter 18 (1999). cited by applicant .
Britton et al., Generation of a recombinant avian coronavirus infectious bronchitis virus using transient dominant selection, J. Virol. Methods, 123(2):203-11 (2005). cited by applicant .
Britton et al., Modification of the avian coronavirus infectious bronchitis virus for vaccine development, Bioeng. Bugs., 3(2):114-9 (2012). cited by applicant .
Casais et al., Recombinant avian infectious bronchitis virus expressing a heterologous spike gene demonstrates that the spike protein is a determinant of cell tropism, J. Virol., 77(16):9084-9 (2003). cited by applicant .
Casais et al., Reverse genetics system for the avian coronavirus infectious bronchitis virus, J. Virol., 75(24):12359-69 (2001). cited by applicant .
Devereux et al., A comprehensive set of sequence analysis programms for the VAX. Nucl. Acids Res.12: 387-95 (1984). cited by applicant .
Cavanagh et al., Manipulation of the infectious bronchitis coronavirus genome for vaccine development and analysis of the accessory proteins, Vaccine, 25(30):5558-62 (2007). cited by applicant .
International Preliminary Report on Patentability, International Application No. PCT/GB2015/052124, dated Jan. 24, 2017. cited by applicant .
International Search Report and Written Opinion, International Application No. PCT/GB2015/052124, dated Oct. 9, 2015. cited by applicant .
Larkin et al., Clustal W and Clustal X version 2.0, Bioinformatics, 23(21):2947-8 (2007). cited by applicant .
Menachery et al., Attenuation and restoration of severe acute respiratory syndrome coronavirus mutant lacking 2'-o-methyltransferase activity, J. Virol., 88(8):4251-64 (2014). cited by applicant .
Tatusova et al., BLAST 2 Sequences, a new tool for comparing protein and nucleotide sequences, FEMS Microbiol. Lett., 174(2):247-50 (1999). cited by applicant .
Wang et al., Attenuation of porcine reproductive and respiratory syndrome virus strain MN184 using chimeric construction with vaccine sequence, Virology, 371(2):418-29 (2008). cited by applicant .
Wei et al., Development and characterization of a recombinant infectious bronchitis virus expressing the ectodomain region of S1 gene of H120 strain, Appl. Microbiol. Biotechnol., 98(4):1727-35 (2014). cited by applicant. |
Primary Examiner: Li; Bao Q
Attorney, Agent or Firm: Marshall, Gerstein & Borun LLP
Claims
The invention claimed is:
1. A live, attenuated coronavirus comprising a variant replicase gene encoding polyproteins comprising a mutation in one or both of non-structural protein(s) nsp-10 and nsp-14, wherein the variant replicase gene encodes a protein comprising an amino acid mutation of Pro to Leu at the position corresponding to position 85 of SEQ ID NO: 6, and/or wherein the variant replicase gene encodes a protein comprising an amino acid mutation of Val to Leu at the position corresponding to position 393 of SEQ ID NO: 7.
2. The coronavirus according to claim 1 wherein the variant replicase gene encodes a protein comprising one or more amino acid mutations selected from: an amino acid mutation of Leu to Ile at the position corresponding to position 183 of SEQ ID NO: 8; and an amino acid mutation of Val to Ile at the position corresponding to position 209 of SEQ ID NO: 9.
3. The coronavirus according to claim 1 wherein the replicase gene encodes a protein comprising the amino acid mutations Val to Leu at the position corresponding to position 393 of SEQ ID NO: 7; Leu to Ile at the position corresponding to position 183 of SEQ ID NO: 8; and Val to Ile at the position corresponding to position 209 of SEQ ID NO: 9.
4. The coronavirus according to claim 1 wherein the replicase gene encodes a protein comprising the amino acid mutations Pro to Leu at the position corresponding to position 85 of SEQ ID NO: 6; Val to Leu at the position corresponding to position 393 of SEQ ID NO: 7; Leu to Ile at the position corresponding to position 183 of SEQ ID NO: 8; and Val to Ile at the position corresponding to position 209 of SEQ ID NO: 9.
5. The coronavirus according to claim 1 wherein the replicase gene comprises at least one nucleotide substitutions selected from: C to Tat nucleotide position 12137; and G to C at nucleotide position 18114; compared to the sequence shown as SEQ ID NO: 1; and optionally, comprises one or more nucleotide substitutions selected from T to A at nucleotide position 19047; and G to A at nucleotide position 20139; compared to the sequence shown as SEQ ID NO: 1.
6. The coronavirus according to claim 1 which is an infectious bronchitis virus (IBV).
7. The coronavirus according to claim 1 which is IBV M41.
8. The coronavirus according to claim 7, which comprises an S protein at least, part of which is from an IBV serotype other than M41.
9. The coronavirus according to claim 8, wherein the S1 subunit is from an IBV serotype other than M41.
10. The coronavirus according to claim 8, wherein the S protein is from an IBV serotype other than M41.
11. The coronavirus according to claim 1 which has reduced pathogenicity compared to a coronavirus expressing a corresponding wild-type replicase, wherein the virus is capable of replicating without being pathogenic to the embryo when administered to an embryonated egg.
12. A variant replicase gene as defined in claim 1.
13. A protein encoded by a variant coronavirus replicase gene according to claim 12.
14. A plasmid comprising a replicase gene according to claim 12.
15. A method for making the coronavirus according to claim 1 which comprises the following steps: (i) transfecting a plasmid according to claim 14 into a host cell; (ii) infecting the host cell with a recombining virus comprising the genome of a coronavirus strain with a replicase gene; (iii) allowing homologous recombination to occur between the replicase gene sequences in the plasmid and the corresponding sequences in the recombining virus genome to produce a modified replicase gene; and (iv) selecting for recombining virus comprising the modified replicase gene.
16. The method according to claim 15, wherein the recombining virus is a vaccinia virus.
17. The method according to claim 15 which also includes the step: (v) recovering recombinant coronavirus comprising the modified replicase gene from the DNA from the recombining virus from step (iv).
18. A cell capable of producing a coronavirus according to claim 1.
19. A vaccine comprising a coronavirus according to claim 1 and a pharmaceutically acceptable carrier.
20. A method for treating and/or preventing a disease in a subject which comprises the step of administering a vaccine according to claim 19 to the subject.
21. The method of claim 20, wherein the disease is infectious bronchitis (IB).
22. The method according to claim 20 wherein the method of administration is selected from the group consisting of; eye drop administration, intranasal administration, drinking water administration, post-hatch injection and in ovo injection.
23. The method according to claim 21 wherein the administration is in ovo vaccination.
24. A method for producing a vaccine according to claim 19, which comprises the step of infecting a cell according to claim 18 with a coronavirus according to claim 1.
25. The coronavirus according to claim 1, further comprising a mutation in one or both of nsp-15 and nsp-16.
Description
FIELD OF THE INVENTION
The present invention relates to an attenuated coronavirus comprising a variant replicase gene, which causes the virus to have reduced pathogenicity. The present invention also relates to the use of such a coronavirus in a vaccine to prevent and/or treat a disease.
BACKGROUND TO THE INVENTION
Avian infectious bronchitis virus (IBV), the aetiological agent of infectious bronchitis (IB), is a highly infectious and contagious pathogen of domestic fowl that replicates primarily in the respiratory tract but also in epithelial cells of the gut, kidney and oviduct. IBV is a member of the Order Nidovirales, Family Coronaviridae, Subfamily Corona virinae and Genus Gammacoronavirus; genetically very similar coronaviruses cause disease in turkeys, guinea fowl and pheasants.
Clinical signs of IB include sneezing, tracheal rales, nasal discharge and wheezing. Meat-type birds have reduced weight gain, whilst egg-laying birds lay fewer eggs and produce poor quality eggs. The respiratory infection predisposes chickens to secondary bacterial infections which can be fatal in chicks. The virus can also cause permanent damage to the oviduct, especially in chicks, leading to reduced egg production and quality; and kidney, sometimes leading to kidney disease which can be fatal.
IBV has been reported to be responsible for more economic loss to the poultry industry than any other infectious disease. Although live attenuated vaccines and inactivated vaccines are universally used in the control of IBV, the protection gained by use of vaccination can be lost either due to vaccine breakdown or the introduction of a new IBV serotype that is not related to the vaccine used, posing a risk to the poultry industry.
Further, there is a need in the industry to develop vaccines which are suitable for use in ovo, in order to improve the efficiency and cost-effectiveness of vaccination programmes. A major challenge associated with in ovo vaccination is that the virus must be capable of replicating in the presence of maternally-derived antibodies against the virus, without being pathogenic to the embryo. Current IBV vaccines are derived following multiple passage in embryonated eggs, this results in viruses with reduced pathogenicity for chickens, so that they can be used as live attenuated vaccines. However such viruses almost always show an increased virulence to embryos and therefore cannot be used for in ova vaccination as they cause reduced hatchability. A 70% reduction in hatchability is seen in some cases.
Attenuation following multiple passage in embryonated eggs also suffers from other disadvantages. It is an empirical method, as attenuation of the viruses is random and will differ every time the virus is passaged, so passage of the same virus through a different series of eggs for attenuation purposes will lead to a different set of mutations leading to attenuation. There are also efficacy problems associated with the process: some mutations will affect the replication of the virus and some of the mutations may make the virus too attenuated. Mutations can also occur in the S gene which may also affect immunogenicity so that the desired immune response is affected and the potential vaccine may not protect against the required serotype. In addition there are problems associated with reversion to virulence and stability of vaccines.
It is important that new and safer vaccines are developed for the control of IBV. Thus there is a need for IBV vaccines which are not associated with these issues, in particular vaccines which may be used for in ovo vaccination.
SUMMARY OF ASPECTS OF THE INVENTION
The present inventors have used a reverse genetics approach in order to rationally attenuate IBV. This approach is much more controllable than random attenuation following multiple passages in embryonated eggs because the position of each mutation is known and its effect on the virus, i.e. the reason for attenuation, can be derived.
Using their reverse genetics approach, the present inventors have identified various mutations which cause the virus to have reduced levels of pathogenicity. The levels of pathogenicity may be reduced such that when the virus is administered to an embryonated egg, it is capable of replicating without being pathogenic to the embryo. Such viruses may be suitable for in ovo vaccination, which is a significant advantage and has improvement over attenuated IBV vaccines produced following multiple passage in embryonated eggs.
Thus in a first aspect, the present invention provides a live, attenuated coronavirus comprising a variant replicase gene encoding polyproteins comprising a mutation in one or more of non-structural protein(s) (nsp)-10, nsp-14, nsp-15 or nsp-16.
The variant replicase gene may encode a protein comprising one or more amino acid mutations selected from the list of: Pro to Leu at position 85 of SEQ ID NO: 6, Val to Leu at position 393 of SEQ ID NO: 7; Leu to Ile at position 183 of SEQ ID NO: 8; Val to Ile at position 209 of SEQ ID NO: 9.
The replicase gene may encode a protein comprising the amino acid mutation Pro to Leu at position 85 of SEQ ID NO: 6.
The replicase gene may encode a protein comprising the amino acid mutations Val to Leu at position 393 of SEQ ID NO: 7; Leu to Ile at position 183 of SEQ ID NO: 8; and Val to Ile at position 209 of SEQ ID NO: 9.
The replicase gene may encodes a protein comprising the amino acid mutations Pro to Leu at position 85 of SEQ ID NO: 6; Val to Leu at position 393 of SEQ ID NO:7; Leu to Ile at position 183 of SEQ ID NO:8; and Val to Ile at position 209 of SEQ ID NO: 9.
The replicase gene may comprise one or more nucleotide substitutions selected from the list of:
C to T at nucleotide position 12137;
G to C at nucleotide position 18114;
T to A at nucleotide position 19047; and
G to A at nucleotide position 20139;
compared to the sequence shown as SEQ ID NO: 1.
The coronavirus may be an infectious bronchitis virus (IBV).
The coronavirus may be IBV M41.
The coronavirus may comprise an S protein at least part of which is from an IBV serotype other than M41.
For example, the S1 subunit or the entire S protein may be from an IBV serotype other than M41.
The coronavirus according to the first aspect of the invention has reduced pathogenicity compared to a coronavirus expressing a corresponding wild-type replicase, such that when the virus is administered to an embryonated egg, it is capable of replicating without being pathogenic to the embryo.
In a second aspect, the present invention provides a variant replicase gene as defined in connection with the first aspect of the invention.
In a third aspect, the present invention provides a protein encoded by a variant coronavirus replicase gene according to the second aspect of the invention.
In a fourth aspect, the present invention provides a plasmid comprising a replicase gene according to the second aspect of the invention.
In a fifth aspect, the present invention provides a method for making the coronavirus according to the first aspect of the invention which comprises the following steps: (i) transfecting a plasmid according to the fourth aspect of the invention into a host cell; (ii) infecting the host cell with a recombining virus comprising the genome of a coronavirus strain with a replicase gene; (iii) allowing homologous recombination to occur between the replicase gene sequences in the plasmid and the corresponding sequences in the recombining virus genome to produce a modified replicase gene; and (iv) selecting for recombining virus comprising the modified replicase gene.
The recombining virus may be a vaccinia virus.
The method may also include the step: (v) recovering recombinant coronavirus comprising the modified replicase gene from the DNA from the recombining virus from step (iv).
In a sixth aspect, the present invention provides a cell capable of producing a coronavirus according to the first aspect of the invention.
In a seventh aspect, the present invention provides a vaccine comprising a coronavirus according to the first aspect of the invention and a pharmaceutically acceptable carrier.
In an eighth aspect, the present invention provides a method for treating and/or preventing a disease in a subject which comprises the step of administering a vaccine according to the seventh aspect of the invention to the subject.
Further aspects of the invention provide: the vaccine according to the seventh aspect of the invention for use in treating and/or preventing a disease in a subject. use of a coronavirus according to the first aspect of the invention in the manufacture of a vaccine for treating and/or preventing a disease in a subject.
The disease may be infectious bronchitis (IB).
The method of administration of the vaccine may be selected from the group consisting of; eye drop administration, intranasal administration, drinking water administration, post-hatch injection and in ovo injection.
Vaccination may be by in ova vaccination.
The present invention also provides a method for producing a vaccine according to the seventh aspect of the invention, which comprises the step of infecting a cell according to the sixth aspect of the invention with a coronavirus according to the first aspect of the invention.
DESCRIPTION OF THE FIGURES
FIG. 1--Growth kinetics of M41-R-6 and M41-R-12 compared to M41-CK (M41 EP4) on CK cells
FIG. 2--Clinical signs, snicking and wheezing, associated with M41-R-6 and M41-R-12 compared to M41-CK (M41 EP4) and Beau-R (Bars show mock, Beau-R, M41-R 6, M41-R 12, M41-CK EP4 from left to right of each timepoint).
FIG. 3--Ciliary activity of the viruses in tracheal rings isolated from tracheas taken from infected chicks. 100% ciliary activity indicates no effect by the virus; apathogenic, 0% activity indicates complete loss of ciliary activity, complete ciliostasis, indicating the virus is pathogenic (Bars show mock, Beau-R, M41-R 6, M41-R 12, M41-CK EP4 from left to right of each timepoint).
FIG. 4--Clinical signs, snicking, associated with M41R-nsp10rep and M41R-nsp14,15,16rep compared to M41-R-12 and M41-CK (M41 EP5) (Bars show mock, M41-R12; M41R-nsp10rep; M41R-nsp14,15,16rep and M41-CK EP5 from left to right of each timepoint).
FIG. 5--Ciliary activity of M41R-nsp10rep and M41R-nsp14,15,16rep compared to M41-R-12 and M41-CK in tracheal rings isolated from tracheas taken from infected chicks (Bars show mock; M41-R12; M41R-nsp10rep; M41R-nsp14,15,16rep and M41-CK EP5 from left to right of each timepoint).
FIG. 6--Clinical signs, snicking, associated with M41R-nsp10, 15rep, M41R-nsp10, 14, 15rep, M41R-nsp10, 14, 16rep, M41R-nsp10, 15, 16rep and M41-K compared to M41-CK (Bars show mock, M41R-nsp10,15rep1; M41R-nsp10,14,16rep4; M41R-nsp10,15,16rep8; M41R-nsp10,14,15rep10; M41-K6 and M41-CK EP4 from left to right of each timepoint).
FIG. 7--Clinical signs, wheezing, associated with M41R-nsp10, 15rep, M41R-nsp10, 14, 15rep, M41R-nsp10, 14, 16rep, M41R-nsp10, 15, 16rep and M41-K compared to M41-CK (Bars show mock, M41R-nsp10,15rep1; M14R-nsp10,14,16rep4; M41R-nsp10,15,16rep8; M41R-nsp10,14,15rep10; M41-K6 and M41-CK EP4 from left to right of each timepoint).
FIG. 8--Ciliary activity of M41R-nsp10, 15rep, M41R-nsp10, 14, 15rep, M41R-nsp10, 14, 16rep, M41R-nsp10, 15, 16rep and M41-K compared to M41-CK in tracheal rings isolated from tracheas taken from infected chicks (Bars show mock, M41R-nsp10,15rep1; M41R-nsp10,14,16rep4; M41R-nsp10,15,16rep8; M41R-nsp10,14,15rep10; M41-K6 and M41-CK EP4 from left to right of each timepoint).
FIG. 9--Growth kinetics of rIBVs compared to M41-CK on CK cells. FIG. 9A shows the results for M41-R and M41-K. FIG. 9B shows the results for M41-nsp10 rep; M41R-nsp14, 15, 16 rep; M41R-nsp10, 15 rep; M41R-nsp10, 15, 16 rep; M41R-nsp10, 14, 15 rep; and M41R-nsp10, 14, 16.
FIG. 10--Position of amino acid mutations in mutated nsp10, nsp14, nsp15 and nsp16 sequences.
FIG. 11--A) Snicking; B) Respiratory symptoms (wheezing and rales combined) and C) Ciliary activity of rIBV M41R-nsp 10,14 rep and rIBV M41R-nsp 10,16 rep compared to M41-CK (Bars show mock, M41R-nsp10,14rep; M41R-nsp10,16rep and M41-K from left to right of each timepoint).
DETAILED DESCRIPTION
The present invention provides a coronavirus comprising a variant replicase gene which, when expressed in the coronavirus, causes the virus to have reduced pathogenicity compared to a corresponding coronavirus which comprises the wild-type replicase gene.
Coronavirus
Gammacoronavirus is a genus of animal virus belonging to the family Coronaviridae. Coronaviruses are enveloped viruses with a positive-sense single-stranded RNA genome and a helical symmetry.
The genomic size of coronaviruses ranges from approximately 27 to 32 kilobases, which is the longest size for any known RNA virus.
Coronaviruses primarily infect the upper respiratory or gastrointestinal tract of mammals and birds. Five to six different currently known strains of coronaviruses infect humans. The most publicized human coronavirus, SARS-CoV which causes severe acute respiratory syndrome (SARS), has a unique pathogenesis because it causes both upper and lower respiratory tract infections and can also cause gastroenteritis. Middle East respiratory syndrome coronavirus (MERS-CoV) also causes a lower respiratory tract infection in humans. Coronaviruses are believed to cause a significant percentage of all common colds in human adults.
Coronaviruses also cause a range of diseases in livestock animals and domesticated pets, some of which can be serious and are a threat to the farming industry. Economically significant coronaviruses of livestock animals include infectious bronchitis virus (IBV) which mainly causes respiratory disease in chickens and seriously affects the poultry industry worldwide; porcine coronavirus (transmissible gastroenteritis, TGE) and bovine coronavirus, which both result in diarrhoea in young animals. Feline coronavirus has two forms, feline enteric coronavirus is a pathogen of minor clinical significance, but spontaneous mutation of this virus can result in feline infectious peritonitis (FIP), a disease associated with high mortality.
There are also two types of canine coronavirus (CCoV), one that causes mild gastrointestinal disease and one that has been found to cause respiratory disease. Mouse hepatitis virus (MHV) is a coronavirus that causes an epidemic murine illness with high mortality, especially among colonies of laboratory mice.
Coronaviruses are divided into four groups, as shown below: Alpha Canine coronavirus (CCoV) Feline coronavirus (FeCoV) Human coronavirus 229E (HCoV-229E) Porcine epidemic diarrhoea virus (PEDV) Transmissible gastroenteritis virus (TGEV) Human Coronavirus NL63 (NL or New Haven) Beta Bovine coronavirus (BCoV) Canine respiratory coronavirus (CRCoV)--Common in SE Asia and Micronesia Human coronavirus OC43 (HCoV-OC43) Mouse hepatitis virus (MHV) Porcine haemagglutinating encephalomyelitis virus (HEV) Rat coronavirus (Roy). Rat Coronavirus is quite prevalent in Eastern Australia where, as of March/April 2008, it has been found among native and feral rodent colonies. (No common name as of yet) (HCoV-HKU1) Severe acute respiratory syndrome coronavirus (SARS-CoV) Middle East respiratory syndrome coronavirus (MERS-CoV) Gamma Infectious bronchitis virus (IBV) Turkey coronavirus (Bluecomb disease virus) Pheasant coronavirus Guinea fowl coronavirus Delta Bulbul coronavirus (BuCoV) Thrush coronavirus (ThCoV) Munia coronavirus (MuCoV) Porcine coronavirus (PorCov) HKU15
The variant replicase gene of the coronavirus of the present invention may be derived from an alphacoronavirus such as TGEV; a betacoronavirus such as MHV; or a gammacoronavirus such as IBV.
As used herein the term "derived from" means that the replicase gene comprises substantially the same nucleotide sequence as the wild-type replicase gene of the relevant coronavirus. For example, the variant replicase gene of the present invention may have up to 80%, 85%, 90%, 95%, 98% or 99% identity with the wild type replicase sequence. The variant coronavirus replicase gene encodes a protein comprising a mutation in one or more of non-structural protein (nsp)-10, nsp-14, nsp-15 or nsp-16 when compared to the wild-type sequence of the non-structural protein.
IBV
Avian infectious bronchitis (IB) is an acute and highly contagious respiratory disease of chickens which causes significant economic losses. The disease is characterized by respiratory signs including gasping, coughing, sneezing, tracheal rales, and nasal discharge. In young chickens, severe respiratory distress may occur. In layers, respiratory distress, nephritis, decrease in egg production, and loss of internal egg quality and egg shell quality are common.
In broilers, coughing and rattling are common clinical signs, rapidly spreading in all the birds of the premises. Morbidity is 100% in non-vaccinated flocks. Mortality varies depending on age, virus strain, and secondary infections but may be up to 60% in non-vaccinated flocks.
The first IBV serotype to be identified was Massachusetts, but in the United States several serotypes, including Arkansas and Delaware, are currently circulating, in addition to the originally identified Massachusetts type.
The IBV strain Beaudette was derived following at least 150 passages in chick embryos. IBV Beaudette is no longer pathogenic for hatched chickens but rapidly kills embryos.
H120 is a commercial live attenuated IBV Massachusetts serotype vaccine strain, attenuated by approximately 120 passages in embryonated chicken eggs. H52 is another Massachusetts vaccine, and represents an earlier and slightly more pathogenic passage virus (passage 52) during the development of H120. Vaccines based on H120 are commonly used.
IB QX is a virulent field isolate of IBV. It is sometimes known as "Chinese QX" as it was originally isolated following outbreaks of disease in the Qingdao region in China in the mid 1990s. Since that time the virus has crept towards Europe. From 2004, severe egg production issues have been identified with a very similar virus in parts of Western Europe, predominantly in the Netherlands, but also reported from Germany, France, Belgium, Denmark and in the UK.
The virus isolated from the Dutch cases was identified by the Dutch Research Institute at Deventer as a new strain that they called D388. The Chinese connection came from further tests which showed that the virus was 99% similar to the Chinese QX viruses. A live attenuated QX-like IBV vaccine strain has now been developed.
IBV is an enveloped virus that replicates in the cell cytoplasm and contains an non-segmented, single-stranded, positive sense RNA genome. IBV has a 27.6 kb RNA genome and like all coronaviruses contains the four structural proteins; spike glycoprotein (S), small membrane protein (E), integral membrane protein (M) and nucleocapsid protein (N) which interacts with the genomic RNA.
The genome is organised in the following manner: 5'UTR--polymerase (replicase) gene--structural protein genes (S-E-M-N)--UTR 3'; where the UTR are untranslated regions (each .about.500 nucleotides in IBV).
The lipid envelope contains three membrane proteins: S, M and E. The IBV S protein is a type I glycoprotein which oligomerizes in the endoplasmic reticulum and is assembled into homotrimer inserted in the virion membrane via the transmembrane domain and is associated through non-covalent interactions with the M protein. Following incorporation into coronavirus particles, the S protein is responsible for binding to the target cell receptor and fusion of the viral and cellular membranes. The S glycoprotein consists of four domains: a signal sequence that is cleaved during synthesis; the ectodomain, which is present on the outside of the virion particle; the transmembrane region responsible for anchoring the S protein into the lipid bilayer of the virion particle; and the cytoplasmic tail.
All coronaviruses also encode a set of accessory protein genes of unknown function that are not required for replication in vitro, but may play a role in pathogenesis. IBV encodes two accessory genes, genes 3 and 5, which both express two accessory proteins 3a, 3b and 5a, 5b, respectively.
The variant replicase gene of the coronavirus of the present invention may be derived from an IBV. For example the IBV may be IBV Beaudette, H120, H52, IB QX, D388 or M41.
The IBV may be IBV M41. M41 is a prototypic Massachusetts serotype that was isolated in the USA in 1941. It is an isolate used in many labs throughout the world as a pathogenic lab stain and can be obtained from ATCC (VR-21.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.
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 ACTTAAGATAGATATTAATATATATCTATCACACTAGCCTTGCGCTAGATTTCCAACTTA ACAAAACGGACTTAAATACCTACAGCTGGTCCTCATAGGTGTTCCATTGCAGTGCACTTT AGTGCCCTGGATGGCACCTGGCCACCTGTCAGGTTTTTGTTATTAAAATCTTATTGTTGC TGGTATCACTGCTTGTTTTGCCGTGTCTCACTTTATACATCCGTTGCTTGGGCTACCTAG TATCCAGCGTCCTACGGGCGCCGTGGCTGGTTCGAGTGCGAAGAACCTCTGGTTCATCTA GCGGTAGGCGGGTGTGTGGAAGTAGCACTTCAGACGTACCGGTTCTGTTGTGTGAAATAC GGGGTCACCTCCCCCCACATACCTCTAAGGGCTTTTGAGCCTAGCGTTGGGCTACGTTCT CGCATAAGGTCGGCTATACGACGTTTGTAGGGGGTAGTGCCAAACAACCCCTGAGGTGAC AGGTTCTGGTGGTGTTTAGTGAGCAGACATACAATAGACAGTGACAACATGGCTTCAAGC CTAAAACAGGGAGTATCTGCGAAACTAAGGGATGTCATTGTTGTATCCAAAGAGATTGCT GAACAACTTTGTGACGCTTTGTTTTTCTATACGTCACACAACCCTAAGGATTACGCTGAT GCTTTTGCAGTTAGGCAGAAGTTTGATCGTAATCTGCAGACTGGGAAACAGTTCAAATTT GAAACTGTGTGTGGTCTCTTCCTCTTGAAGGGAGTTGACAAAATAACACCTGGCGTCCCA GCAAAAGTCTTAAAAGCCACTTCTAAGTTGGCAGATTTAGAAGACATCTTTGGTGTCTCT CCCTTTGCAAGAAAATATCGTGAACTTTTGAAGACAGCATGCCAGTGGTCTCTTACTGTA GAAACACTGGATGCTCGTGCACAAACTCTTGATGAAATTTTTGACCCTACTGAAATACTT TGGCTTCAGGTGGCAGCAAAAATCCAAGTTTCGGCTATGGCGATGCGCAGGCTTGTTGGA GAAGTAACTGCAAAAGTCATGGATGCTTTGGGCTCAAATATGAGTGCTCTTTTCCAGATT TTTAAACAACAAATAGTCAGAATTTTTCAAAAAGCGCTGGCTATTTTTGAGAATGTGAGT GAATTACCACAGCGTATTGCAGCACTTAAGATGGCTTTTGCTAAGTGTGCCAAGTCCATT ACTGTTGTGGTTATGGAGAGGACTCTAGTTGTTAGAGAGTTCGCAGGAACTTGTCTTGCA AGCATTAATGGTGCTGTTGCAAAATTCTTTGAAGAACTCCCAAATGGTTTCATGGGTGCT AAAATTTTCACTACACTTGCCTTCTTTAGGGAGGCTGCAGTGAAAATTGTGGATAACATA CCAAATGCACCGAGAGGCACTAAAGGGTTTGAAGTCGTTGGTAATGCCAAAGGTACACAA GTTGTTGTGCGTGGCATGGGAAATGACTTAACACTGGTTGAGCAAAAAGCTGAAATTGCT GTGGAGTCAGAAGGTTGGTCTGCAATTTTGGGTGGACATCTTTGCTATGTCTTTAAGAGT GGTGATCGCTTTTACGCGGCACCTCTTTCAGGAAATTTTGCATTGCATGATGTGCATTGT TGTGAGCGTGTTGTCTGTCTTTCTGATGGTGTAACACCGGAGATAAATGATGGACTTATT CTTGCAGCAATCTACTCTTCTTTTAGTGTCGCAGAACTTGTGGCAGCCATTAAAAGGGGT GAACCATTTAAGTTTCTGGGTCATAAATTTGTGTATGCAAAGGATGCAGCAGTTTCTTTT ACATTAGCGAAGGCTGCTACTATTGCAGATGTTTTGAAGCTGTTTCAATCAGCGCGTGTG AAAGTAGAAGATGTTTGGTCTTCACTTACTGAAAAGTCTTTTGAATTCTGGAGGCTTGCA TATGGAAAAGTGCGTAATCTCGAAGAATTTGTTAAGACTTGTTTTTGTAAGGCTCAAATG GCGATTGTGATTTTAGCGACAGTGCTTGGAGAGGGCATTTGGCATCTTGTTTCGCAAGTC ATCTATAAAGTAGGTGGTCTTTTTACTAAAGTTGTTGACTTTTGTGAAAAATATTGGAAA GGTTTTTGTGCACAGTTGAAAAGAGCTAAGCTCATTGTCACTGAAACCCTCTGTGTTTTG AAAGGAGTTGCACAGCATTGTTTTCAACTATTGCTGGATGCAATACAGTTTATGTATAAA AGTTTTAAGAAGTGTGCACTTGGTAGAATCCATGGAGACTTGCTCTTCTGGAAAGGAGGT GTGCACAAAATTATTCAAGAGGGCGATGAAATTTGGTTTGAGGGCATTGATAGTATTGAT GTTGAAGATCTGGGTGTTGTTCAAGAAAAATTGATTGATTTTGATGTTTGTGATAATGTG ACACTTCCAGAGAACCAACCCGGTCATATGGTTCAAATCGAGGATGACGGAAAGAACTAC ATGTTCTTCCGCTTCAAAAAGGATGAGAACATTTATTATACACCAATGTCACAGCTTGGT GCTATTAATGTGGTTTGCAAAGCAGGCGGTAAAACTGTCACCTTTGGAGAAACTACTGTG CAAGAAATACCACCACCTGATGTTGTGTTTATTAAGGTTAGCATTGAGTGTTGTGGTGAA CCATGGAATACAATCTTCAAAAAGGCTTATAAGGAGCCCATTGAAGTAGAGACAGACCTC ACAGTTGAACAATTGCTCTCTGTGGTCTATGAGAAAATGTGTGATGATCTCAAGCTGTTT CCGGAGGCTCCAGAACCACCACCATTTGAGAATGTCACACTTGTTGATAAGAATGGTAAA GATTTGGATTGCATAAAATCATGCCATCTGATCTATCGTGATTATGAGAGCGATGATGAC ATCGAGGAAGAAGATGCAGAAGAATGTGACACGGATTCAGGTGATGCTGAGGAGTGTGAC ACTAATTCAGAATGTGAAGAAGAAGATGAGGATACTAAAGTGTTGGCTCTTATACAAGAC CCGGCAAGTAACAAATATCCTCTGCCTCTTGATGATGATTATAGCGTCTACAATGGATGT ATTGTTCATAAGGACGCTCTCGATGTTGTGAATTTACCATCTGGTGAAGAAACCTTTGTT GTCAATAACTGCTTTGAAGGGGCTGTTAAAGCTCTTCCGCAGAAAGTTATTGATGTTCTA GGTGACTGGGGTGAGGCTGTTGATGCGCAAGAACAATTGTGTCAACAAGAATCAACTCGG GTCATATCTGAGAAATCAGTTGAGGGTTTTACTGGTAGTTGTGATGCAATGGCTGAACAA GCTATTGTTGAAGAGCAGGAAATAGTACCTGTTGTTGAACAAAGTCAGGATGTAGTTGTT TTTACACCTGCAGACCTAGAAGTTGTTAAAGAAACAGCAGAAGAGGTTGATGAGTTTATT CTCATTTCTGCTGTCCCTAAAGAAGAAGTTGTGTCTCAGGAGAAAGAGGAGCCACAGGTT GAGCAAGAGCCTACCCTAGTTGTTAAAGCACAACGTGAGAAGAAGGCTAAAAAGTTCAAA GTTAAACCAGCTACATGTGAAAAACCCAAATTTTTGGAGTACAAAACATGTGTGGGTGAT TTGGCTGTTGTAATTGCCAAAGCATTGGATGAGTTTAAAGAGTTCTGCATTGTAAACGCT GCAAATGAGCACATGTCGCATGGTGGTGGCGTTGCAAAGGCAATTGCAGACTTTTGTGGA CCGGACTTTGTTGAATATTGCGCGGACTATGTTAAGAAACATGGTCCACAGCAAAAACTT GTCACACCTTCATTTGTTAAAGGCATTCAATGTGTGAATAATGTTGTAGGACCTCGCCAT GGAGACAGCAACTTGCGTGAGAAGCTTGTTGCTGCTTACAAGAGTGTTCTTGTAGGTGGA GTGGTTAACTATGTTGTGCCAGTTCTCTCATCAGGGATTTTTGGTGTAGATTTTAAAATA TCAATAGATGCTATGCGCGAAGCTTTTAAAGGTTGTGCCATACGCGTTCTTTTATTTTCT CTGAGTCAAGAACACATCGATTATTTCGATGCAACTTGTAAGCAGAAGACAATTTATCTT ACGGAGGATGGTGTTAAATACCGCTCTGTTGTTTTAAAACCTGGTGATTCTTTGGGTCAA TTTGGACAGGTTTTTGCAAGAAATAAGGTAGTCTTTTCGGCTGATGATGTTGAGGATAAA GAAATCCTCTTTATACCCACAACTGACAAGACTATTCTTGAATATTATGGTTTAGATGCG CAAAAGTATGTAACATATTTGCAAACGCTTGCGCAGARATGGGATGTTCAATATAGAGAC AATTTTGTTATATTAGAGTGGCGTGACGGAAATTGCTGGATTAGTTCAGCAATAGTTCTC CTTCAAGCTGCTAAAATTAGATTTAAAGGTTTTCTTGCAGAAGCATGGGCTAAACTGTTG GGTGGAGATCCTACAGACTTTGTTGCCTGGTGTTATGCAAGTTGCAATGCTAAAGTAGGT GATTTTTCAGATGCTAATTGGCTTTTGGCCAATTTAGCAGAACATTTTGACGCAGATTAC ACAAATGCACTTCTTAAGAAGTGTGTGTCGTGCAATTGTGGTGTTAAGAGTTATGAACTT AGGGGTCTTGAAGCCTGTATTCAGCCAGTTCGAGCACCTAATCTTCTACATTTTAAAACG CAATATTCAAATTGCCCAACCTGTGGTGCAAGTAGTACGGATGAAGTAATAGAAGCTTCA TTACCGTACTTATTGCTTTTTGCTACTGATGGTCCTGCTACAGTTGATTGTGATGAAAAT GCTGTAGGGACTGTTGTTTTCATTGGCTCTACTAATAGTGGCCATTGTTATACACAAGCC GATGGTAAGGCTTTTGACAATCTTGCTAAGGATAGAAAATTTGGAAGGAAGTCGCCTTAC ATTACAGCAATGTATACACGTTTTTCTCTTAGGAGTGAAAATCCCCTACTTGTTGTTGAA CATAGTAAGGGTAAAGCTAAAGTAGTAAAAGAAGATGTTTCTAACCTTGCTACTAGTTCT AAAGCCAGTTTTGACGATCTTACTGACTTTGAACACTGGTATGATAGCAACATCTATGAG AGTCTTAAAGTGCAGGAGACACCTGATAATCTTGATGAATATGTGTCATTTACGACAAAG GAAGATTCTAAGTTGCCACTGACACTTAAAGTTAGAGGTATCAAATCAGTTGTTGACTTT AGGTCTAAGGATGGTTTTACTTATAAGTTAACACCTGATACTGATGAAAATTCAAAAACA CCAGTCTACTACCCAGTCTTGGATTCTATTAGTCTTAGGGCAATATGGGTTGAAGGCAGT GCTAATTTTGTTGTTGGGCATCCAAATTATTATAGTAAGTCTCTCCGAATTCCCACGTTT TGGGAAAATGCCGAGAGCTTTGTTAAAATGGGTTATAAAATTGATGGTGTAACTATGGGC CTTTGGCGTGCAGAACACCTTAATAAACCTAATTTGGAGAGAATTTTTAACATTGCTAAG AAAGCTATTGTTGGATCTAGTGTTGTTACTACGCAGTGTGGTAAAATACTAGTTAAAGCA GCTACATACGTTGCCGATAAAGTAGGTGATGGTGTAGTTCGCAATATTACAGATAGAATT AAGGGTCTTTGTGGATTCACACGTGGCCATTTTGAAAAGAAAATGTCCCTACAATTTCTA AAGACACTTGTGTTCTTTTTCTTTTATTTCTTAAAGGCTAGTGCTAAGAGTTTAGTTTCT AGCTATAAGATTGTGTTATGTAAGGTGGTGTTTGCTACCTTACTTATAGTGTGGTTTATA TACACAAGTAATCCAGTAGTGTTTACTGGAATACGTGTGCTAGACTTCCTATTTGAAGGT TCTTTATGTGGTCCTTATAATGACTACGGTAAAGATTCTTTTGATGTGTTACGGTATTGT GCAGGTGATTTTACTTGTCGTGTGTGTTTACATGATAGAGATTCACTTCATCTGTACAAA CATGCTTATAGCGTAGAACAAATTTATAAGGATGCAGCTTCTGGCATTAACTTTAATTGG AATTGGCTTTATTTGGTCTTTCTAATATTATTTGTTAAGCCAGTGGCAGGTTTTGTTATT ATTTGTTATTGTGTTAAGTATTTGGTATTGAGTTCAACTGTGTTGCAAACTGGTGTAGGT TTTCTAGATTGGTTTGTAAAAACAGTTTTTACCCATTTTAATTTTATGGGAGCGGGATTT TATTTCTGGCTCTTTTACAAGATATACGTACAAGTGCATCATATATTGTACTGTAAGGAT GTAACATGTGAAGTGTGCAAGAGAGTTGCACGCAGCAACAGGCAAGAGGTTAGCGTTGTA GTTGGTGGACGCAAGCAAATAGTGCATGTTTACACTAATTCTGGCTATAACTTTTGTAAG AGACATAATTGGTATTGTAGAAATTGTGATGATTATGGTCACCAAAATACATTTATGTCC CCTGAAGTTGCTGGCGAGCTTTCTGAAAAGCTTAAGCGCCATGTTAAACCTACAGCATAT GCTTACCACGTTGTGTATGAGGCATGCGTGGTTGATGATTTTGTTAATTTAAAATATAAG GCTGCAATTGCTGGTAAGGATAATGCATCTTCTGCTGTTAAGTGTTTCAGTGTTACAGAT TTTTTAAAGAAAGCTGTTTTTCTTAAGGAGGCATTGAAATGTGAACAAATATCTAATGAT GGTTTTATAGTGTGTAATACACAGAGTGCGCATGCACTAGAGGAAGCAAAGAATGCAGCC GTCTATTATGCGCAATATCTGTGTAAGCCAATACTTATACTTGACCAGGCACTTTATGAG CAATTAATAGTAGAGCCTGTGTCTAAGAGTGTTATAGATAAAGTGTGTAGCATTTTGTCT AATATAATATCTGTAGATACTGCAGCTTTAAATTATAAGGCAGGCACACTTCGTGATGCT CTGCTTTCTATTACTAAAGACGAAGAAGCCGTAGATATGGCTATCTTCTGCCACAATCAT GAAGTGGAATACACTGGTGACGGTTTTACTAATGTGATACCGTCATATGGTATGGACACT GATAAGTTGACACCTCGTGATAGAGGGTTTTTGATAAATGCAGATGCTTCTATTGCTAAT TTAAGAGTCAAAAATGCTCCTCCGGTAGTATGGAAGTTTTCTGATCTTATTAAATTGTCT GACAGTTGCCTTAAATATTTAATTTCAGCTACTGTCAAGTCAGGAGGTCGTTTCTTTATA ACAAAGTCTGGTGCTAAACAAGTTATTTCTTGTCATACCCAGAAACTGTTGGTAGAGAAA AAGGCAGGTGGTGTTATTAATAACACTTTTAAATGGTTTATGAGTTGTTTTAAATGGCTT TTTGTCTTTTATATACTTTTTACAGCATGTTGTTTGGGTTACTACTATATGGAGATGAAT
AAAAGTTTTGTTCACCCCATGTATGATGTAAACTCCACACTGCATGTTGAAGGGTTCAAA GTTATAGACAAAGGTGTTATTAGAGAGATTGTGTCAGAAGATAATTGTTTCTCTAATAAG TTTGTTAATTTTGACGCCTTTTGGGGTAAATCATATGAAAATAATAAAAACTGTCCAATT GTTACAGTTGTTATAGATGGTGACGGGACAGTAGCTGTTGGTGTTCCTGGTTTTGTATCA TGGGTTATGGATGGTGTTATGTTTGTGCATATGACACAGACTGATCGTAGACCTTGGTAC ATTCCTACCTGGTTTAATAGAGAAATTGTTGGTTACACTCAGGATTCAATTATCACTGAG GGTAGTTTTTATACATCTATAGCATTATTTTCTGCTAGATGTTTATATTTAACAGCCAGC AATACACCTCAATTGTATTGTTTTAATGGCGACAATGATGCACCTGGAGCCTTACCATTT GGTAGTATTATTCCTCATAGAGTATACTTCCAACCTAATGGTGTTAGGCTTATAGTTCCA CAACAAATACTGCATACACCCTACATAGTGAAGTTTGTTTCAGACAGCTATTGTAGAGGT AGTGTATGTGAGTATACTAAACCAGGTTACTGTGTGTCACTAGACTCCCAATGGGTTTTG TTTAATGATGAATACATTAGTAAACCTGGCGTTTTCTGTGGTTCTACTGTTAGAGAACTT ATGTTTAATATGGTTAGTACATTCTTTACTGGTGTCAACCCTAATATTTATATTCAGCTA GCAACTATGTTTTTAATACTAGTTGTTATTGTGTTAATTTTTGCAATGGTTATAAAGTTT CAAGGTGTTTTTAAAGCTTATGCGACCATTGTGTTTACAATAATGTTAGTTTGGGTTATT AATGCATTTGTTTTGTGTGTACATAGTTATAATAGTGTTTTAGCTGTTATATTATTAGTA CTCTATTGCTATGCATCATTGGTTACAAGTCGCAATACTGCTATAATAATGCATTGTTGG CTTGTTTTTACCTTTGGTTTAATAGTACCCACATGGTTGGCTTGTTGCTATCTGGGATTT ATTCTTTATATGTACACACCGTTGGTTTTCTGGTGTTACGGTACTACTAAAAATACTCGT AAGTTGTATGATGGCAACGAGTTTGTTGGTAATTATGACCTTGCTGCGAAGAGCACTTTT GTTATTCGTGGTACTGAATTTGTTAAGCTTACGAATGAGATAGGTGATAAATTTGAAGCC TATCTTTCTGCGTATGCTAGACTTAAATACTATTCAGGCACTGGTAGTGAGCAAGATTAC TTGCAAGCTTGTCGTGCATGGTTAGCTTATGCTTTGGACCAATATAGAAATAGTGGTGTT GAGGTTGTTTATACCCCACCGCGTTACTCTATTGGTGTTAGTAGACTACACGCTGGTTTT AAAAAACTAGTTTCTCCTAGTAGTGCTGTTGAGAAGTGCATTGTTAGTGTCTCTTATAGA GGCAATAATCTTAATGGACTGTGGCTGGGTGATTCTATTTACTGCCCACGCCATGTGTTA GGTAAGTTTAGTGGTGACCAGTGGGGTGACGTACTAAACCTTGCTAATAATCATGAGTTT GAAGTTGTAACTCAAAATGGTGTTACTTTGAATGTTGTCAGCAGGCGGCTTAAAGGAGCA GTTTTAATTTTACAAACTGCAGTTGCCAATGCTGAAACTCCTAAGTATAAGTTTGTTAAA GCTAATTGTGGTGATAGTTTCACTATAGCTTGTTCTTATGGTGGTACAGTTATAGGACTT TACCCTGTCACTATGCGTTCTAATGGTACTATTAGAGCATCTTTCCTAGCAGGAGCCTGT GGCTCAGTTGGTTTTAATATAGAAAAGGGTGTAGTTAATTTCTTTTATATGCACCATCTT GAGTTACCTAATGCATTACACACTGGAACTGACCTAATGGGTGAGTTTTATGGTGGTTAT GTAGATGAAGAGGTTGCGCAAAGAGTGCCACCAGATAATCTAGTTACTAACAATATTGTA GCATGGCTCTATGGGGCAATTATTAGTGTTAAAGAAAGTAGTTTTTCACAACCTAAATGG TTGGAGAGTACTACTGTTTCTATTGAAGATTACAATAGGTGGGCTAGTGATAATGGTTTT ACTCCATTTTCCACTAGTACTGCTATTACTAAATTAAGTGCTATAACTGGGGTTGATGTT TGTAAACTCCTTCGCACTATTATGGTAAAAAGTGCTCAATGGGGTAGTGATCCCATTTTA GGACAATATAATTTTGAAGACGAATTGACACCAGAATCTGTATTTAATCAAGTTGGTGGT GTTAGGTTACAGTCTTCTTTTGTAAGAAAAGCTACATCTTGGTTTTGGAGTAGATGTGTA TTAGCTTGCTTCTTGTTTGTGTTGTGTGCTATTGTCTTATTTACGGCAGTGCCACTTAAG TTTTATGTACATGCAGCTGTTATTTTGTTGATGGCTGTGCTCTTTATTTCTTTTACTGTT AAACATGTTATGGCATACATGGACACTTTCCTATTGCCTACATTGATTACAGTTATTATT GGAGTTTGTGCTGAAGTCCCTTTCATATACAATACTCTAATTAGTCAAGTTGTTATTTTC TTAAGCCAATGGTATGATCCTGTAGTCTTTGATACTATGGTACCATGGATGTTATTGCCA TTAGTGTTGTACACTGCTTTTAAGTGTGTACAAGGCTGCTATATGAATTCTTTCAATACT TCTTTGTTAATGCTGTATCAGTTTATGAAGTTAGGTTTTGTTATTTACACCTCTTGAAAC ACTCTTACTGCATATACAGAAGGTAATTGGGAGTTATTCTTTGAGTTGGTTCACACTATT GTGTTGGCTAATGTTAGTAGTAATTCCTTAATTGGTTTAATTGTTTTTAAGTGTGCTAAG TGGATTTTATATTATTGCAATGCAACATACTTTAATAATTATGTGTTAATGGCAGTCATG GTTAATGGCATAGGCTGGCTTTGCACCTGTTACTTTGGATTGTATTGGTGGGTTAATAAA GTTTTTGGTTTAACCTTAGGTAAATACAATTTTAAAGTTTCAGTAGATCAATATAGGTAT ATGTGTTTGCATAAGGTAAATCCACCTAAAACTGTGTGGGAGGTCTTTACTACAAATATA CTTATACAAGGAATTGGAGGCGATCGTGTGTTGCCTATAGCTACAGTGCAATCTAAATTG AGTGATGTAAAGTGTACAACTGTTGTTTTAATGCAGCTTTTGACTAAGCTTAATGTTGAA GCAAATTCAAAAATGCATGCTTATCTTGTTGAGTTACACAATAAAATCCTCGCATCTGAT GATGTTGGAGAGTGCATGGATAATTTATTGGGTATGCTTATAACACTATTTTGTATAGAT TCTACTATTGATTTGGGTGAGTATTGTGATGATATACTTAAGAGGTCAACTGTATTACAA TCGGTTACTCAAGAGTTTTCGCACATACCCTCGTATGCTGAATATGAAAGAGCTAAGAGT ATTTATGAAAAGGTTTTAGCCGATTCTAAAAATGGTGGTGTAACACAGCAAGAGCTTGCT GCATATCGTAAAGCTGCCAATATTGCAAAGTCAGTTTTTGATAGAGACTTGGCTGTTCAA AAGAAGTTAGATAGCATGGCAGAACGTGCTATGACAACAATGTATAAAGAGGCGCGTGTA ACTGATAGAAGAGCAAAATTAGTTTCATCATTACATGCACTACTTTTTTCAATGCTTAAG AAAATAGATTCTGAGAAGCTTAATGTCTTATTTGACCAGGCGAATAGTGGTGTTGTACCC CTAGCAACTGTTCCAATTGTTTGTAGTAATAAGCTTACCCTTGTTATACCAGACCCAGAG ACGTGGGTCAAGTGTGTGGAGGGTGTGCATGTTACATATTCAACAGTTGTTTGGAATATA GACTGTGTTACTGATGCCGATGGCACAGAGTTACACCCCACTTCTACAGGTAGTGGATTG ACTTACTGTATAAGTGGTGATAATATAGCATGGCCTTTAAAGGTTAACTTGACTAGGAAT GGGCATAATAAGGTTGATGTTGCCTTGCAAAATAATGAGCTTATGCCTCACGGTGTAAAG ACAAAGGCTTGCGTAGCAGGTGTAGATCAAGCACATTGTAGCGTTGAGTCTAAATGTTAT TATACAAGTATTAGTGGCAGTTCAGTTGTAGCTGCTATTACCTCTTCAAATCCTAATCTG AAAGTAGCCTCTTTTTTGAATGAGGCAGGTAATCAGATTTATGTAGACTTAGACCGAGCA TGTAAATTTGGTATGAAAGTGGGTGATAAGGTTGAAGTTGTTTACCTGTATTTTATAAAA AATACGAGGTCTATTGTAAGAGGTATGGTACTTGGTGCTATATCTAATGTTGTTGTGTTA CAATCTAAAGGTCATGAGACAGAGGAAGTGGATGCTGTAGGCATTCTCTCACTTTGTTCT TTTGCAGTAGATCCTGCGGATACATATTGTAAATATGTGGCAGCAGGTAATCAACCTTTA GGTAACTGTGTTAAAATGTTGACAGTACATAATGGTAGTGGTTTTGCAATAACATCAAAG CCAAGTCCAACTCCGGATCAGGATTCTTATGGAGGAGCTTCTGTGTGTCTTTATTGTAGA GCACATATAGCACACCCTGGCGGAGCAGGAAATTTAGATGGACGCTGTCAATTTAAAGGT TCTTTTGTGCAAATACCTACTACGGAGAAAGATCCTGTTGGATTCTGTCTACGTAACAAG GTTTGCACTGTTTGTCAGTGTTGGATTGGTTATGGATGTCAGTGTGATTCACTTAGACAA CCTAAACCTTCTGTTCAGTCAGTTGCTGTTGCATCTGGTTTTGATAAGAATTATTTAAAC GGGTACGGGGTAGCAGTGAGGCTCGGCTGATACCCCTAGCTAATGGATGTGACCCCGATG TTGTAAAGCGAGCCTTTGATGTTTGTAATAAGGAATCAGCCGGTATGTTTCAAAATTTGA AGCGTAACTGTGCACGATTCCAAGAAGTACGTGATACTGAAGATGGAAATCTTGAGTATT GTGATTCTTATTTTGTGGTTAAACAAACCACTCCTAGTAATTATGAACATGAGAAAGCTT GTTATGAAGACTTAAAGTCAGAAGTAACAGCTGATCATGATTTCTTTGTGTTCAATAAGA ACATTTATAATATTAGTAGGCAGAGGCTTACTAAGTATACTATGATGGATTTTTGCTATG CTTTGCGGCACTTTGACCCAAAGGATTGCGAAGTTCTTAAAGAAATACTTGTCACTTATG GTTGTATAGAAGATTATCACCCTAAGTGGTTTGAAGAGAATAAGGATTGGTACGACCCAA TAGAAAACCCTAAATATTATGCCATGTTGGCTAAAATGGGACCTATTGTACGAGGTGCTT TATTGAATGCTATTGAGTTCGGAAACCTCATGGTTGAAAAAGGTTATGTTGGTGTTATTA CACTTGATAACCAAGATCTTAATGGCAAATTTTATGATTTTGGTGATTTTCAGAAGACAG CGCCTGGTGCTGGTGTTCCTGTTTTTGATACGTATTATTCTTACATGATGCCCATCATAG CCATGACTGATGCGTTGGCACCTGAGAGGTATTTTGAATATGATGTGCATAAGGGTTATA AATCTTATGATCTCCTCAAGTATGATTATACTGAGGAGAAACAAGATTTGTTTCAGAAGT ACTTTAAGTATTGGGATCAAGAGTATCACCCTAACTGTCGCGACTGTAGTGATGACAGGT GTTTGATACATTGTGCAAACTTCAACATCTTGTTTTCTACACTTGTACCGCAGACTTCTT TCGGTAATTTGTGTAGAAAGGTTTTTGTTGATGGTGTACCATTTATAGCTACTTGTGGCT ATCATTCTAAGGAACTTGGTGTTATTATGAATCAAGATAACACCATGTCATTTTCAAAAA TGGGTTTGAGTGAACTCATGGAGTTTGTTGGAGATCGTGGCTTGTTAGTGGGGACATGCA ATAAATTAGTGGATCTTAGAACGTCTTGTTTTAGTGTTTGTGCTTTAGCGTCTGGTATTA CTCATCAAACGGTAAAACCAGGTCACTTTAACAAGGATTTCTACGATTTTGCAGAGAAGG CTGGTATGTTTAAGGAAGGTTCTTCTATACCACTTAAACATTTCTTCTACCCACAGACTG GTAATGCTGCTATAAACGATTATGATTATTATCGTTATAACAGGCCTACCATGTTTGATA TACGTCAACTTTTATTTTGTTTAGAAGTGACTTCTAAATATTTTGAATGTTATGAAGGCG GCTGTATACCAGCAAGCCAAGTTGTAGTTAACAATTTAGATAAGAGTGCAGGTTATCCGT TCAATAAGTTTGGAAAGGCCCGTCTCTATTATGAAATGAGTCTAGAGGAGCAGGACCAAC TCTTTGAGAGTACAAAGAAGAACGTCCTGCCTACTATAACTCAGATGAATTTAAAATATG CCATATCCGCGAAAAATAGAGCGCGTACAGTGGCAGGTGTGTCTATCCTTTCTACTATGA CTAATAGGCAGTTTCATCAGAAGATTCTTAAGTCTATAGTCAACACTAGAAACGCTCCTG TAGTTATTGGAACAACCAAGTTTTATGGCGGTTGGGATAACATGTTGAGAAACCTTATTC AGGGTGTTGAAGACCCGATTCTTATGGGTTGGGATTATCCAAAGTGTGATAGAGCAATGC CTAATTTGTTGCGTATAGCAGCATCTTTAGTACTCGCTCGTAAACACACTAATTGTTGTA CTTGGTCTGAACGCGTTTATAGGTTGTATAATGAATGCGCTCAGGTTTTATCTGAAACTG TCTTAGCTACAGGTGGTATATATGTGAAACCTGGTGGTACTAGCAGTGGAGATGCTACTA CTGCTTATGCAAACAGTGTTTTCAACATAATACAAGCCACATCTGCTAATGTTGCGCGTC TTTTGAGTGTTATAACGCGTGATATTGTATATGATGACATTAAGAGCTTGCAGTATGAAT TGTACCAGCAGGTTTATAGGCGAGTCAATTTTGACCCAGCATTTGTTGAAAAGTTTTATT CTTATTTGTGTAAGAATTTCTCATTGATGATCTTGTCTGACGACGGTGTTGTTTGTTATA ACAACACATTAGCCAAACAAGGTCTTGTAGCAGATATTTCTGGTTTTAGAGAAGTTCTCT ACTATCAGAACAATGTTTTTATGGCTGATTCTAAATGTTGGGTTGAACCAGATTTAGAAA AAGGCCCACATGAATTTTGTTCACAGCACACAATGTTAGTGGAGGTTGATGGTGAGCCTA GATACTTGCCATATCCAGACCCATCACGTATTTTGTGTGCATGTGTTTTTGTAGATGATT TGGATAAGACAGAATCTGTGGCTGTTATGGAGCGTTATATCGCTCTTGCCATAGATGCGT ACCCACTAGTACATCATGAAAATGAGGAGTACAAGAAGGTATTCTTTGTGCTTCTTTCAT
ACATCAGAAAACTCTATCAAGAGCTTTCTCAGAATATGCTTATGGACTACTCTTTTGTAA TGGATATAGATAAGGGTAGTAAATTTTGGGAACAGGAGTTCTATGAAAATATGTATAGAG CCCCTACAACATTACAGTGTTGTGGCGTTTGTGTAGTGTGTAATAGTCAAACTATATTGC GCTGTGGTAATTGTATTCGCAAACCATTTTTGTGTTGTAAGTGTTGCTATGACCATGTCA TGCACACAGACCACAAAAATGTTTTGTCTATAAATCCTTACATTTGCTCACAGCCAGGTT GTGGTGAAGCAGATGTTACTAAATTGTACCTCGGAGGTATGTCATACTTCTGCGGTAATC ATAAACCAAAGTTATCAATACCGTTAGTATCTAATGGTACAGTGTTTGGAATTTACAGGG CTAATTGTGCAGGTAGCGAAAATGTTGATGATTTTAATCAACTAGCTACTACTAATTGGT CTACTGTGGAACCTTATATTTTGGCAAATCGTTGTGTAGATTCGTTGAGACGCTTTGCTG CAGAGACAGTAAAAGCTACAGAAGAATTACATAAGCAACAATTTGCTAGTGCAGAAGTGA GAGAAGTACTCTCAGATCGTGAATTGATTCTGTCTTGGGAGCCAGGTAAAACCAGGCCTC CATTGAATAGAAATTATGTTTTCACTGGCTTTCACTTTACTAGAACTAGTAAAGTTCAGC TCGGTGATTTTACATTTGAAAAAGGTGAAGGTAAGGACGTTGTCTATTATCGAGCGACGT CTACTGCTAAATTGTCTGTTGGAGACATTTTTGTTTTAACCTCACACAATGTTGTTTCTC TTATAGCGCCAACGTTGTGTCCTCAGCAAACCTTTTCTAGGTTTGTGAATTTAAGACCTA ATGTGATGGTACCTGCGTGTTTTGTAAATAACATTCCATTGTACCATTTAGTAGGCAAGC AGAAGCGTACTACAGTACAAGGCCCTCCTGGCAGTGGTAAATCCCATTTTGCTATAGGAT TGGCGGCTTACTTTAGTAACGCCCGTGTCGTTTTTACTGCATGCTCTCATGCAGCTGTTG ATGCTTTATGTGAAAAAGCTTTTAAGTTTCTTAAAGTAGATGATTGCACTCGTATAGTAC CTCAAAGGACTACTATCGATTGCTTCTCTAAGTTTAAAGGTAATGACACAGGCAAAAAGT ACATTTTTAGTACTATTAATGCCTTGCCAGAAGTTAGTTGTGACATTCTTTTGGTTGACG AGGTTAGTATGTTGACCAATTACGAATTGTCTTTTATTAATGGTAAGATAAACTATCAAT ATGTTGTGTATGTAGGTGATCCTGCTCAATTACCGGCGCCTCGTACGTTGCTTAACGGTT CACTCTCTCCAAAGGATTATAATGTTGTCACAAACCTTATGGTTTGTGTTAAACCTGACA TTTTCCTTGCAAAGTGTTACCGTTGTCCTAAAGAAATTGTAGATACTGTTTCTACTCTTG TATATGATGGAAAGTTTATTGCAAATAACCCGGAATCACGTCAGTGTTTCAAGGTTATAG TTAATAATGGTAATTCTGATGTAGGACATGAAAGTGGCTCAGCCTACAACATAACTCAAT TAGAATTTGTGAAAGATTTTGTCTGTCGCAATAAGGAATGGCGGGAAGCAACATTCATTT CACCTTATAATGCTATGAACCAGAGAGCCTACCGTATGCTTGGACTTAATGTTCAGACAG TAGACTCGTCTCAAGGTTCGGAGTATGATTATGTTATCTTTTGTGTTACTGCAGATTCGC AGCATGCACTGAATATTAACAGATTCAATGTAGCGCTTACAAGAGCCAAGCGTGGTATAC TAGTTGTCATGCGTCAGCGTGATGAACTATATTCAGCTCTTAAGTTTATAGAGCTTGATA GTGTAGCAAGTCTGCAAGGTACAGGCTTGTTTAAAATTTGCAACAAAGAGTTTAGTGGTG TTCACCCAGCTTATGCAGTCACAACTAAGGCTCTTGCTGCAACTTATAAAGTTAATGATG AACTTGCTGCACTTGTTAACGTGGAAGCTGGTTCAGAAATAACATATAAACATCTTATTT CTTTGTTAGGGTTTAAGATGAGTGTTAATGTTGAAGGCTGCCACAACATGTTTATAACAC GTGATGAGGCTATCCGCAACGTAAGAGGTTGGGTAGGTTTTGATGTAGAAGCAACACATG CTTGCGGTACTAACATTGGTACTAACCTGCCTTTCCAAGTAGGTTTCTCTACTGGTGCAG ACTTTGTAGTTACGCCTGAGGGACTTGTAGATACTTCAATAGGCAATAATTTTGAGCCTG TGAATTCTAAAGCACCTCCAGGTGAACAATTTAATCACTTGAGAGCGTTATTCAAAAGTG CTAAACCTTGGCATGTTGTAAGGCCAAGGATTGTGCAAATGTTAGCGGATAACCTGTGCA ACGTTTCAGATTGTGTAGTGTTTGTCACGTGGTGTCATGGCCTAGAACTAACCACTTTGC GCTATTTTGTTAAAATAGGCAAGGACCAAGTTTGTTCTTGCGGTTCTAGAGCAACAACTT TTAATTCTCATACTCAGGCTTATGCTTGTTGGAAGCATTGCTTGGGTTTTGATTTTGTTT ATAATCCACTCTTAGTGGATATTCAACAGTGGGGTTATTCTGGTAACCTACAATTTAACC ATGATTTGCATTGTAATGTGCATGGACACGCACATGTAGCTTCTGCGGATGCTATTATGA CGCGTTGTCTTGCAATTAATAATGCATTTTGTCAAGATGTCAACTGGGATTTAACTTACC CTCATATAGCAAATGAGGATGAAGTCAATTCTAGCTGTAGATATTTACAACGCATGTATC TTAATGCATGTGTTGATGCTCTTAAAGTTAACGTTGTCTATGATATAGGCAACCCTAAAG GTATAAAATGTGTTAGACGTGGAGACTTAAATTTTAGATTCTATGATAAGAATCCAATAG TACCCAATGTCAAGCAGTTTGAGTATGACTATAATCAGCACAAAGATAAGTTTGCTGATG GTCTTTGTATGTTTTGGAATTGTAATGTGGATTGTTATCCCGACAATTCCTTAGTTTGTA GGTACGACACACGAAATTTGAGTGTGTTTAACCTACCTGGTTGTAATGGTGGTAGCTTGT ATGTTAACAAGCATGCATTCCACACACCTAAATTTGATCGCACTAGCTTTCGTAATTTGA AAGCTATGCCATTCTTTTTCTATGACTCATCGCCTTGCGAGACCATTCAATTGGATGGAG TTGCGCAAGACCTTGTGTCATTAGCTACGAAAGATTGTATCACAAAATGCAACATAGGCG GTGCTGTTTGTAAAAAGCACGCACAAATGTATGCAGATTTTGTGACTTCTTATAATGCAG CTGTTACTGCTGGTTTTACTTTTTGGGTTACTAATAATTTTAACCCATATAATTTGTGGA AAAGTTTTTCAGCTCTCCAGTCTATCGACAATATTGCTTATAATATGTATAAGGGTGGTC ATTATGATGCTATTGCAGGAGAAATGCCCACTATCGTAACTGGAGATAAAGTTTTTGTTA TAGATCAAGGCGTAGAAAAAGCAGTTTTTTTTAATCAAACAATTCTGCCTAGATCTGTAG CGTTTGAGCTGTATGCGAAGAGAAATATTCGCACACTGCCAAACAACCGTATTTTGAAAG GTTTGGGTGTAGATGTGACTAATGGATTTGTAATTTGGGATTACACGAACCAAACACCAC TATACCGTAATACTGTTAAGGTATGTGCATATACAGACATAGAACCAAATGGCCTAATAG TGCTGTATGATGATAGATATGGTGATTACCAGTCTTTTCTAGCTGCTGATAATGCTGTTT TAGTTTCTACACAGTGTTACAAGCGGTATTCGTATGTAGAAATACCGTCAAACCTGCTTG TTCAGAACGGTATTCCGTTAAAAGATGGAGCGAACCTGTATGTTTATAAGCGTGTTAATG GTGCGTTTGTTACGCTACCTAACACATTAAACACACAGGGTCGCAGTTATGAAACTTTTG AACCTCGTAGTGATGTTGAGCGTGATTTTCTCGACATGTCTGAGGAGAGTTTTGTAGAAA AGTATGGTAAAGAATTAGGTCTACAGCACATACTGTATGGTGAAGTTGATAAGCCCCAAT TAGGTGGTTTACACACTGTTATAGGTATGTGCAGACTTTTACGTGCGAATAAGTTGAACG CAAAGTCTGTTACTAATTCTGATTCTGATGTCATGCAAAATTATTTTGTATTGGCAGACA ATGGTTCCTACAAGCAAGTGTGTACTGTTGTGGATTTGCTGCTTGATGATTTCTTAGAAC TTCTTAGGAACATACTGAAAGAGTATGGTACTAATAAGTCTAAAGTTGTAACAGTGTCAA TTGATTACCATAGCATAAATTTTATGACTTGGTTTGAAGATGGCATTATTAAAACATGTT ATCCACAGCTTCAATCAGCATGGACGTGTGGTTATAATATGCCTGAACTTTATAAAGTTC AGAATTGTGTTATGGAACCTTGCAACATTCCTAATTATGGTGTTGGAATAGCGTTGCCAA GTGGTATTATGATGAATGTGGCAAAGTATACACAACTCTGTCAATACCTTTCGAAAACAA CAATGTGTGTACCGCATAATATGCGAGTAATGCATTTTGGAGCTGGAAGTGACAAAGGAG TGGCTCCAGGTAGTACTGTTCTTAAACAATGGCTCCCAGAAGGGACACTCCTTGTCGATA ATGATATTGTAGACTATGTGTCTGATGCACATGTTTCTGTGCTTTCAGATTGCAATAAAT ATAAGACAGAGCACAAGTTTGATCTTGTGATATCTGATATGTATACAGACAATGATTCAA AAAGAAAGCATGAAGGCGTGATAGCCAATAATGGCAATGATGACGTTTTCATATATCTCT CAAGTTTTCTTCGTAATAATTTGGCTCTAGGTGGTAGTTTTGCTGTAAAAGTGACAGAGA CAAGTTGGCACGAAGTTTTATATGACATTGCACAGGATTGTGCATGGTGGACAATGTTTT GTACAGCAGTGAATGCCTCTTCTTCAGAAGCATTCTTGGTTGGTGTTAATTATTTGGGTG CAAGTGAAAAGGTTAAGGTTAGTGGAAAAACGCTGCACGCAAATTATATATTTTGGAGGA ATTGTAATTATTTACAAACCTCTGCTTATAGTATATTTGACGTTGCTAAGTTTGATTTGA GATTGAAAGCAACACCAGTTGTTAATTTGAAAACTGAACAAAAGAGAGACTTAGTGTTTA ATTTAATTAAGTGTGGTAAGTTACTGGTAAGAGATGTTGGTAACACCTCTTTTACTAGTG TACCAAAGTGCCTTTAGACCACCTAATGGTTGGCATTTACACGGGGGTGCTTATGCGGTA GTTAATATTTCTAGCGAATCTAATAATGCAGGCTCTTCACCTGGGTGTATTGTTGGTACT ATTCATGGTGGTCGTGTTGTTAATGCTTCTTCTATAGCTATGACGGCACCGTCATCAGGT ATGGCTTGGTCTAGCAGTCAGTTTTGTACTGCACACTGTAACTTTTCAGATACTACAGTG TTTGTTACACATTGTTATAAATATGATGGGTGTCCTATAACTGGCATGCTTCAAAAGAAT TTTTTACGTGTTTCTGCTATGAAAAATGGCCAGCTTTTCTATAATTTAACAGTTAGTGTA GCTAAGTACCCTACTTTTAAATCATTTCAGTGTGTTAATAATTTAACATCCGTATATTTA AATGGTGATCTTGTTTACACCTCTAATGAGACCACAGATGTTACATCTGCAGGTGTTTAT TTTAAAGCTGGTGGACCTATAACTTATAAAGTTATGAGAGAAGTTAAAGCCCTGGCTTAT TTTGTTAATGGTACTGCACAAGATGTTATTTTGTGTGATGGATCACCTAGAGGCTTGTTA GCATGCCAGTATAATACTGGCAATTTTTCAGATGGCTTTTATCCTTTTATTAATAGTAGT TTAGTTAAGCAGAAGTTTATTGTCTATCGTGAAAATAGTGTTAATACTACTTTTACGTTA CACAATTTCACTTTTCATAATGAGACTGGCGCCAACCCTAATCCTAGTGGTGTTCAGAAT ATTCAAACTTACCAAACACAAACAGCTCAGAGTGGTTATTATAATTTTAATTTTTCCTTT CTGAGTAGTTTTGTTTATAAGGAGTCTAATTTTATGTATGGATCTTATCACCCAAGTTGT AATTTTAGACTAGAAACTATTAATAATGGCTTGTGGTTTAATTCACTTTCAGTTTCAATT GCTTACGGTCCTCTTCAAGGTGGTTGCAAGCAATCTGTCTTTAGTGGTAGAGCAACTTGT TGTTATGCTTATTCATATGGAGGTCCTTCGCTGTGTAAAGGTGTTTATTCAGGTGAGTTA GATCTTAATTTTGAATGTGGACTGTTAGTTTATGTTACTAAGAGCGGTGGCTCTCGTATA CAAACAGCCACTGAACCGCCAGTTATAACTCGACACAATTATAATAATATTACTTTAAAT ACTTGTGTTGATTATAATATATATGGCAGAACTGGCCAAGGTTTTATTACTAATGTAACC GACTCAGCTGTTAGTTATAATTATCTAGCAGACGCAGGTTTGGCTATTTTAGATACATCT GGTTCCATAGACATCTTTGTTGTACAAGGTGAATATGGTCTTACTTATTATTAGGTTAAC CCTTGCGAAGATGTCAACCAGCAGTTTGTAGTTTCTGGTGGTAAATTAGTAGGTATTCTT ACTTCACGTAATGAGACTGGTTCTCAGCTTCTTGAGAACCAGTTTTACATTAAAATCACT AATGGAACACGTCGTTTTAGACGTTCTATTACTGAAAATGTTGGAAATTGCCCTTATGTT AGTTATGGTAAGTTTTGTATAAAACCTGATGGTTCAATTGCCACAATAGTACCAAAACAA TTGGAACAGTTTGTGGCACCTTTACTTAATGTTACTGAAAATGTGCTCATACCTAACAGT TTTAATTTAACTGTTACAGATGAGTACATACAAACGCGTATGGATAAGGTCCAAATTAAT TGTCTGCAGTATGTTTGTGGCAATTCTCTGGATTGTAGAGATTTGTTTCAACAATATGGG CCTGTTTGTGACAACATATTGTCTGTAGTAAATAGTATTGGTCAAAAAGAAGATATGGAA CTTTTGAATTTCTATTCTTCTACTAAACCGGCTGGTTTTAATACACCATTTCTTAGTAAT GTTAGCACTGGTGAGTTTAATATTTCTCTTCTGTTAACAACTCCTAGTAGTCCTAGAAGG CGTTCTTTTATTGAAGACCTTCTATTTACAAGCGTTGAATCTGTTGGATTACCAACAGAT GACGCATACAAAAATTGCACTGCAGGACCTTTAGGTTTTCTTAAGGACCTTGCGTGTGCT
CGTGAATATAATGGTTTGCTTGTGTTGCCTCCCATTATAACAGCAGAAATGCAAATTTTG TATACTAGTTCTCTAGTAGCTTCTATGGCTTTTGGTGGTATTACTGCAGCTGGTGCTATA CCTTTTGCCACACAACTGCAGGCTAGAATTAATCACTTGGGTATTACCCAGTCACTTTTG TTGAAGAATCAAGAAAAAATTGCTGCTTCCTTTAATAAGGCCATTGGTCGTATGCAGGAA GGTTTTAGAAGTACATCTCTAGCATTACAACAAATTCAAGATGTTGTTAATAAGCAGAGT GCTATTCTTACTGAGACTATGGCATCACTTAATAAAAATTTTGGTGCTATTTCTTCTATG ATTCAAGAAATCTACCAGCAACTTGACGCCATACAAGCAAATGCTCAAGTGGATCGTCTT ATAACTGGTAGATTGTCATCACTTTCTGTTTTAGCATCTGCTAAGCAGGCGGAGCATATT AGAGTGTCACAACAGCGTGAGTTAGCTACTCAGAAAATTAATGAGTGTGTTAAGTCACAG TCTATTAGGTACTCCTTTTGTGGTAATGGACGACATGTTCTAACCATACCGCAAAATGCA CCTAATGGTATAGTGTTTATACACTTTTCTTATACTCCAGATAGTTTTGTTAATGTTACT GCAATAGTGGGTTTTTGTGTAAAGCCAGCTAATGCTAGTCAGTATGCAATAGTACCCGCT AATGGTAGGGGTATTTTTATACAAGTTAATGGTAGTTACTACATCACAGCACGAGATATG TATATGCCAAGAGCTATTACTGCAGGAGATATAGTTACGCTTACTTCTTGTCAAGCAAAT TATGTAAGTGTAAATAAGACCGTCATTACTACATTCGTAGACAATGATGATTTTGATTTT AATGACGAATTGTCAAAATGGTGGAATGACACTAAGCATGAGCTACCAGACTTTGACAAA TTCAATTACACAGTACCTATACTTGACATTGATAGTGAAATTGATCGTATTCAAGGCGTT ATACAGGGTCTTAATGACTCTTTAATAGACCTTGAAAAACTTTCAATACTCAAAACTTAT ATTAAGTGGCCTTGGTATGTGTGGTTAGCCATAGCTTTTGCCACTATTATCTTCATCTTA ATACTAGGATGGGTTTTCTTCATGACTGGATGTTGTGGTTGTTGTTGTGGATGCTTTGGC ATTATGCCTCTAATGAGTAAGTGTGGTAAGAAATCTTCTTATTACACGACTTTTGATAAC GATGTGGTAACTTAACAATACAGACCTAAAAAGTCTGTTTAATGATTCAAAGTCCCACGT CCTTCCTAATAGTATTAATTTTTCTTTGGTGTAAACTTGTACTAAGTTGTTTTAGAGAGT TTATTATAGCGCTCCAACAACTAATACAAGTTTTACTCCAAATTATCAATAGTAACTTAC AGCCTAGACTGACCCTTTGTCACAGTCTAGACTAATGTTAAACTTAGAAGCAATTATTGA AACTGGTGAGCAAGTGATTCAAAAAATCAGTTTCAATTTACAGCATATTTCAAGTGTATT AAACACAGAAGTATTTGACCCCTTTGACTATTGTTATTACAGAGGAGGTAATTTTTGGGA AATAGAGTCAGCTGAAGATTGTTCAGGTGATGATGAATTTATTGAATAAGTCGCTAGAGG AAAATGGAAGTTTTCTAACAGCGCTTTATATATTTGTAGGATTTTTAGCACTTTATCTTC TAGGTAGAGCACTTCAAGCATTTGTACAGGCTGCTGATGCTTGTTGTTTATTTTGGTATA CATGGGTAGTAATTCCAGGAGCTAAGGGTACAGCCTTTGTATATAAGTATACATATGGTA GAAAACTTAACAATCGGGAATTAGAAGCAGTTATTGTCAACGAGTTTCCTAAGAACGGTT GGAATAATAAAAATCCAGCAAATTTTCAAGATGTCCAACGAGACAAATTGTACTCTTGAC TTTGAACAGTCAGTTGAGCTTTTTAAAGAGTATAATTTATTTATAACTGCATTCTTGTTG TTCTTAACCATAATACTTCAGTATGGCTATGCAACAAGAAGTAAGTTTATTTATATACTG AAAATGATAGTGTTATGGTGCTTTTGGCCCCTTAACATTGCAGTAGGTGTAATTTCATGT ATATACCCACCAAACACAGGAGGTCTTGTCGCAGCGATAATACTTACAGTGTTTGCGTGT CTGTCTTTTGTAGGTTATTGGATCCAGAGTATTAGACTCTTTAAGCGGTGTAGGTCATGG TGGTCATTTAACCCAGAATCTAATGCCGTAGGTTCAATACTCCTAACTAATGGTCAACAA TGTAATTTTGCTATAGAGAGTGTGCCAATGGTGCTTTCTCCAATTATAAAGAATGGTGTT CTTTATTGTGAGGGTCAGTGGCTTGCTAAGTGTGAACCAGACCACTTGCCTAAAGATATA TTTGTTTGTACACCGGATAGACGTAATATCTACCGTATGGTGCAGAAATATACTGGTGAC CAAAGCGGAAATAAGAAACGGTTTGCTACGTTTGTCTATGCAAAGCAGTCAGTAGATACT GGCGAGCTAGAAAGTGTAGCAACAGGAGGGAGTAGTCTTTACACCTAAATGTGTGTGTGT AGAGAGTATTTAAAATTATTCTTTAATAGTGCCTCTATTTTAAGAGCGCATAATAGTATT ATTTTTGAGGATATTAATATAAATCCTCTCTGTTTTATACTCTCTTTTCAAGAGCTATTA TTTAAAAAACAGTTTTTCCACTCTTTTGTGCCAAAAACTATTGTTGTTAATGGTGTAACC TTTCAAGTAGATAATGGAAAAGTCTACTACGAAGGAAAACCAATTTTTCAGAAAGGTTGT TGTAGGTTGTGGTTGAGTTATAAAAAAGATTAAACTACCTACTACACTTATTTTTATAAG AGGCGTTTTATCTTACAAGCGCTTAATAAATACGGACGATGAAATGGCTGACTAGTTTTG TAAGGGCAGTTATTTCATGTTATAAACCCCTATTATTAACTCAATTAAGAGTATTAGATA GGTTAATCTTAGATCATGGACCAAAACACATCTTAACGTGTGTTAGGTGCGTGATTTTGT TTCAATTAGATTTAGTTTATAGGTTGGCGTATACGCCTACTCAATCGCTGGTATGAATAA TAGTAAAGATAATCCTTTTTGCGGAGCAATAGCAAGAAAAGCGCGAATTTATCTGAGAGA AGGATTAGATTGTGTTTACTTTCTTAACAAAGCAGGACAAGCAGAGTCTTGTCCCGCGTG TACCTCTCTAGTATTCCAGGGGAAAACTTGTGAGGAACACAAATATAATAATAATCTTTT GTCATGGCAAGCGGTAAGGCAACTGGAAAGACAGATGCCCCAGCTCCAGTCATCAAACTA GGAGGACCAAAGCCACCTAAAGTTGGTTCTTCTGGAAATGTATCTTGGTTTCAAGCAATA AAAGCCAAGAAGTTAAATTCACCTCCGCCTAAGTTTGAAGGTAGCGGTGTTCCTGATAAT GAAAATCTAAAACCAAGTCAGCAGCATGGATATTGGAGACGCCAAGCTAGGTTTAAGCCA GGTAAAGGTGGAAGAAAACCAGTCCCAGATGCTTGGTATTTTTAGTATACTGGAACAGGA CCAGCCGCTAACCTGAATTGGGGTGATAGCCAAGATGGTATAGTGTGGGTTGCTGGTAAG GGTGCTGATACTAAATTTAGATCTAATCAGGGTACTCGTGACTCTGACAAGTTTGACCAA TATCCGCTACGGTTTTCAGACGGAGGACCTGATGGTAATTTCCGTTGGGATTTCATTCCT CTGAATCGTGGCAGGAGTGGGAGATCAACAGCAGCTTCATCAGCAGCATCTAGTAGAGCA CCATCACGTGAAGTTTCGCGTGGTCGCAGGAGTGGTTCTGAAGATGATCTTATTGCTCGT GCAGCAAGGATAATTCAGGATCAGCAGAAGAAGGGTTCTCGCATTACAAAGGCTAAGGCT GATGAAATGGCTCACCGCCGGTATTGCAAGCGCAGTATTCCACCTAATTATAAGGTTGAT CAAGTGTTTGGTCCCCGTACTAAAGGTAAGGAGGGAAATTTTGGTGATGACAAGATGAAT GAGGAAGGTATTAAGGATGGGCGCGTTACAGCAATGCTCAACCTAGTTCCTAGCAGCCAT GCTTGTCTTTTCGGAAGTAGAGTGACGCCCAGACTTCAACCAGATGGGCTGCACTTGAAA TTTGAATTTACTACTGTGGTCCCACGTGATGATCCGCAGTTTGATAATTATGTAAAAATT TGTGATCAGTGTGTTGATGGTGTAGGAACACGTCCAAAAGATGATGAACCAAGACCAAAG TCACGCTCAAGTTCAAGACCTGCAACAAGAGGAAATTCTCCAGCGCCAAGACAGCAGCGC CCTAAGAAGGAGAAAAAGCCAAAGAAGCAGGATGATGAAGTGGATAAAGCATTGACCTCA GATGAGGAGAGGAACAATGCACAGCTGGAATTTGATGATGAACCCAAGGTAATTAACTGG GGGGATTCAGCGCTAGGAGAGAATGAACTTTGAGTAAAATTGAATAGTAAGAGTTAAGGA AGATAGGCATGTAGCTTGATTACCTACATGTCTATCGCCAGGGAAATGTCTAATTTGTCT ACTTAGTAGCCTGGAAACGAACGGTAGACCCTTAGATTTTAATTTAGTTTAATTTTTAGT TTAGTTTAAGTTAGTTTAGAGTAGGTATAAAGATGCCAGTGGCGGGGCCACGCGGAGTAC GACCGAGGGTACAGCACTAGGACGCCCATTAGGGGAAGAGCTAAATTTTAGTTTAAGTTA AGTTTAATTGGCTATGTATAGTTAAAATTTATAGGCTAGTATAGAGTTAGAGCAAAAAAA AAAAAAAAAAAAAAAAAAAA
Replicase
In addition to the structural and accessory genes, two-thirds of a coronavirus genome comprises the replicase gene (at the 5' end of the genome), which is expressed as two polyproteins, pp1a and pp1ab, in which pp1ab is an extension product of pp1a as a result of a -1 ribosomal shift mechanism. The two polyproteins are cleaved by two types of virus-encoded proteinases usually resulting in 16 non-structural proteins (Nsp1-16); IBV lacks Nsp1 thereby encoding Nsp2-16.
Thus Gene 1 in IBV encodes 15 (16 in other coronaviruses) non-structural proteins (nsp2-16), which are associated with RNA replication and transcription.
The term `replicase protein` is used herein to refer to the pp1a and pp1ab polyproteins or individual nsp subunits.
The term `replicase gene` is used herein to refer to a nucleic acid sequence which encodes for replicase proteins.
A summary of the functions of coronavirus nsp proteins is provided in Table 1.
TABLE-US-00002 TABLE 1 Nsp Protein Key features 1 Conserved within but not between coronavirus genetic groups; potential regulatory functions in the host cell. 2 Dispensable for MHV and SARS-CoV replication in tissue culture 3 Acidic domain; macro domain with ADRP and poly (ADP-ribose)-binding activities; one or two ZBD- containing papain-like proteases; Y domain 4 Transmembrane domain 5 3C-like main protease, homodimer 6 Transmembrane domain 7 Interacts with nsp8 to form a hexadecamer complex 8 Noncannonical RNA polymerase; interacts with nsp7 to form a hexadecameric complex 9 ssRNA-binding protein, dimer 10 RNA-binding protein, homododecamer, zinc-binding domain, known to interact with nsp14 and nsp16 11 Unknown 12 RNA-dependent RNA polymerase 13 Zinc-binding domain, NTPase, dNTPase, 5'-to-3' RNA and DNA helicase, RNA 5'-triphosphate 14 3'-to 5' exoribonuclease, zinc-binding domain and N7- methyltransferase 15 Uridylate-specific endoribonuclease, homohexamer 16 Putative ribose-2'-O-methyltransferase
The variant replicase gene encoded by the coronavirus of the present invention comprises a mutation in one or more of the sections of sequence encoding nsp-10, nsp-14, nsp-15 or nsp-16.
Nsp10 has RNA-binding activity and appears to be involved in homo and/or heterotypic interactions within other nsps from the pp1a/pp1ab region. It adopts an .alpha./.beta. fold comprised of five .alpha.-helices, one 3.sub.10-helix and three .beta.-strands. Two zinc-binding sites have been identified that are formed by conserved cysteine residues and one histidine residue (Cys-74/Cys-77/His-83/Cys-90; Cys-117/Cys-120/Cys-128/Cys-130). The protein has been confirmed to bind single-stranded and double-stranded RNA and DNA without obvious specificity. Nsp-10 can be cross-linked with nsp-9, suggesting the existing of a complex network of protein-protein interactions involving nsp-7, -8, -9 and -10. In addition, nsp-10 is known to interact with nsp-14 and nsp-16.
Nsp-14 comprises a 3'-to-5' exoribonuclease (ExoN) active domain in the amino-terminal region. SARS-CoV ExoN has been demonstrated to have metal ion-dependent 3'-to-5' exoribonuclease activity that acts on both single-stranded and double-stranded RNA, but not on DNA. Nsp-14 has been shown to have proof-reading activity. This nsp has also been shown to have N7-methyltransferase (MT) activity in the carboxyl-terminal region.
Nsp-15 associated NendoU (nidoviral endoribonuclease, specific for U) RNase activity has been reported for a number of coronaviruses, including SARS-CoV, MHV and IBV. The activities were consistently reported to be significantly enhanced by Mn.sup.2+ ions and there was little activity in the presence of Mg.sup.2+ and Ca.sup.2+. NendoU cleaves at the 3' side of uridylate residues in both single-stranded and double-stranded RNA. The biologically relevant substrate(s) of coronavirus NendoUs remains to be identified.
Nsp-16 has been predicted to mediate ribose-2'-O-methyltransferase (2'-O-MTase) activity and reverse-genetics experiments have shown that the 2'-O-MTase domain is essential for viral RNA synthesis in HCoV-229E and SARS-CoV. The enzyme may be involved in the production of the cap 1 structures of coronavirus RNAs and it may also cooperate with NendoU and ExoN in other RNA processing pathways. 2'-O-MTase might also methylate specific RNAs to protect them from NendoU-mediated cleavage.
The genomic and protein sequences for nsp-10, -14, -15 and -16 are provided as SEQ ID NO: 2-5 and 6-9, respectively.
TABLE-US-00003 (nsp-10 nucleotide sequence- nucleotides 11884-12318 of SEQ ID NO: 1) SEQ ID NO: 2 TCTAAAGGTCATGAGACAGAGGAAGTGGATGCTGTAGGCATTCTCTCACTTTGTTCTTTTGCAGTA GATCCTGCGGATACATATTGTAAATATGTGGCAGCAGGTAATCAACCTTTAGGTAACTGTGTTAAA ATGTTGACAGTACATAATGGTAGTGGTTTTGCAATAACATCAAAGCCAAGTCCAACTCCGGATCAG GATTCTTATGGAGGAGCTTCTGTGTGTCTTTATTGTAGAGCACATATAGCACACCTTGGCGGAGCA GGAAATTTAGATGGACGCTGTCAATTTAAAGGTTCTTTTGTGCAAATACCTACTACGGAGAAAGAT CCTGTTGGATTCTGTCTACGTAACAAGGTTTGCACTGTTTGTCAGTGTTGGATTGGTTATGGATGT CAGTGTGATTCACTTAGACAACCTAAACCTTCTGTTCAG (nsp-14 nucleotide sequence- nucleotides 16938-18500 of SEQ ID NO: 1) SEQ ID NO: 3 GGTACAGGCTTGTTTAAAATTTGCAACAAAGAGTTTAGTGGTGTTCACCCAGCTTATGCAGTCACA ACTAAGGCTCTTGCTGCAACTTATAAAGTTAATGATGAACTTGCTGCACTTGTTAACGTGGAAGCT GGTTCAGAAATAACATATAAACATCTTATTTCTTTGTTAGGGTTTAAGATGAGTGTTAATGTTGAA GGCTGCCACAACATGTTTATAACACGTGATGAGGCTATCCGCAACGTAAGAGGTTGGGTAGGTTTT GATGTAGAAGCAACACATGCTTGCGGTACTAACATTGGTACTAACCTGCCTTTCCAAGTAGGTTTC TCTACTGGTGCAGACTTTGTAGTTACGCCTGAGGGACTTGTAGATACTTCAATAGGCAATAATTTT GAGCCTGTGAATTCTAAAGCACCTCCAGGTGAACAATTTAATCACTTGAGAGCGTTATTCAAAAGT GCTAAACCTTGGCATGTTGTAAGGCCAAGGATTGTGCAAATGTTAGCGGATAACCTGTGCAACGTT TCAGATTGTGTAGTGTTTGTCACGTGGTGTCATGGCCTAGAACTAACCACTTTGCGCTATTTTGTT AAAATAGGCAAGGACCAAGTTTGTTCTTGCGGTTCTAGAGCAACAACTTTTAATTCTCATACTCAG GCTTATGCTTGTTGGAAGCATTGCTTGGGTTTTGATTTTGTTTATAATCCACTCTTAGTGGATATT CAACAGTGGGGTTATTCTGGTAACCTACAATTTAACCATGATTTGCATTGTAATGTGCATGGACAC GCACATGTAGCTTCTGCGGATGCTATTATGACGCGTTGTCTTGCAATTAATAATGCATTTTGTCAA GATGTCAACTGGGATTTAACTTACCCTCATATAGCAAATGAGGATGAAGTCAATTCTAGCTGTAGA TATTTACAACGCATGTATCTTAATGCATGTGTTGATGCTCTTAAAGTTAACGTTGTCTATGATATA GGCAACCCTAAAGGTATTAAATGTGTTAGACGTGGAGACTTAAATTTTAGATTCTATGATAAGAAT CCAATAGTACCCAATGTCAAGCAGTTTGAGTATGACTATAATCAGCACAAAGATAAGTTTGCTGAT GGTCTTTGTATGTTTTGGAATTGTAATGTGGATTGTTATCCCGACAATTCCTTACTTTGTAGGTAC GACACACGAAATTTGAGTGTGTTTAACCTACCTGGTTGTAATGGTGGTAGCTTGTATGTTAACAAG CATGCATTCCACACACCTAAATTTGATCGCACTAGCTTTCGTAATTTGAAAGCTATGCCATTCTTT TTCTATGACTCATCGCCTTGCGAGACCATTCAATTGGATGGAGTTGCGCAAGACCTTGTGTCATTA GCTACGAAAGATTGTATCACAAAATGCAACATAGGCGGTGCTGTTTGTAAAAAGCACGCACAAATG TATGCAGATTTTGTGACTTCTTATAATGCAGCTGTTACTGCTGGTTTTACTTTTTGGGTTACTAAT AATTTTAACCCATATAATTTGTGGAAAAGTTTTTCAGCTCTCCAG (nsp-15 nucleotide sequence- nucleotides 18501-19514 of SEQ ID NO: 1) SEQ ID NO: 4 TCTATCGACAATATTGCTTATAATATGTATAAGGGTGGTCATTATGATGCTATTGCAGGAGAAATG CCCACTATCGTAACTGGAGATAAAGTTTTTGTTATAGATCAAGGCGTAGAAAAAGCAGTTTTTTTT AATCAAACAATTCTGCCTACATCTGTAGCGTTTGAGCTGTATGCGAAGAGAAATATTCGCACACTG CCAAACAACCGTATTTTGAAAGGTTTGGGTGTAGATGTGACTAATGGATTTGTAATTTGGGATTAC ACGAACCAAACACCACTATACCGTAATACTGTTAAGGTATGTGCATATACAGACATAGAACCAAAT GGCCTAATAGTGCTGTATGATGATAGATATGGTGATTACCAGTCTTTTCTAGCTGCTGATAATGCT GTTTTAGTTTCTACACAGTGTTACAAGCGGTATTCGTATGTAGAAATACCGTCAAACCTGCTTGTT CAGAACGGTATTCCGTTAAAAGATGGAGCGAACCTGTATGTTTATAAGCGTGTTAATGGTGCGTTT GTTACGCTACCTAACACAATAAACACACAGGGTCGAAGTTATGAAACTTTTGAACCTCGTAGTGAT GTTGAGCGTGATTTTCTCGACATGTCTGAGGAGAGTTTTGTAGAAAAGTATGGTAAAGAATTAGGT CTACAGCACATACTGTATGGTGAAGTTGATAAGCCCCAATTAGGTGGTTTCCACACTGTTATAGGT ATGTGCAGACTTTTACGTGCGAATAAGTTGAACGCAAAGTCTGTTACTAATTCTGATTCTGATGTC ATGCAAAATTATTTTGTATTGGCAGACAATGGTTCCTACAAGCAAGTGTGTACTGTTGTGGATTTG CTGCTTGATGATTTCTTAGAACTTCTTAGGAACATACTGAAAGAGTATGGTACTAATAAGTCTAAA GTTGTAACAGTGTCAATTGATTACCATAGCATAAATTTTATGACTTGGTTTGAAGATGGCATTATT AAAACATGTTATCCACAGCTTCAA (nsp-16 nucleotide sequence- nucleotides 19515-20423 of SEQ ID NO: 1) SEQ ID NO: 5 TCAGCATGGACGTGTGGTTATAATATGCCTGAACTTTATAAAGTTCAGAATTGTGTTATGGAACCT TGCAACATTCCTAATTATGGTGTTGGAATAGCGTTGCCAAGTGGTATTATGATGAATGTGGCAAAG TATACACAACTCTGTCAATACCTTTCGAAAACAACAATGTGTGTACCGCATAATATGCGAGTAATG CATTTTGGAGCTGGAAGTGACAAAGGAGTGGTGCCAGGTAGTACTGTTCTTAAACAATGGCTCCCA GAAGGGACACTCCTTGTCGATAATGATATTGTAGACTATGTGTCTGATGCACATGTTTCTGTGCTT TCAGATTGCAATAAATATAAGACAGAGCACAAGTTTGATCTTGTGATATCTGATATGTATACAGAC AATGATTCAAAAAGAAAGCATGAAGGCGTGATAGCCAATAATGGCAATGATGACGTTTTCATATAT CTCTCAAGTTTTCTTCGTAATAATTTGGCTCTAGGTGGTAGTTTTGCTGTAAAAGTGACAGAGACA AGTTGGCACGAAGTTTTATATGACATTGCACAGGATTGTGCATGGTGGACAATGTTTTGTACAGCA GTGAATGCCTCTTCTTCAGAAGCATTCTTGATTGGTGTTAATTATTTGGGTGCAAGTGAAAAGGTT AAGGTTAGTGGAAAAACGCTGCACGCAAATTATATATTTTGGAGGAATTGTAATTATTTACAAACC TCTGCTTATAGTATATTTGACGTTGCTAAGTTTGATTTGAGATTGAAAGCAACGCCAGTTGTTAAT TTGAAAACTGAACAAAAGACAGACTTAGTCTTTAATTTAATTAAGTGTGGTAAGTTACTGGTAAGA GATGTTGGTAACACCTCTTTTACTAGTGACTCTTTTGTGTGTACTATGTAG (nsp-10 amino acid sequence) SEQ ID NO: 6 SKGHETEEVDAVGILSLCSFAVDPADTYCKYVAAGNQPLGNCVKMLTVKNGSGFAITSKPSPTPDQ DSYGGASVCLYCRAHIAHPGGAGNLDGRCQFKGSFVQIPTTEKDPVGFCLRNKVCTVCQCWIGYGC QCDSLRQPKPSVQ (nsp-14 amino acid sequence) SEQ ID NO: 7 GTGLFKICNKEFSGVHPAYAVTTKALAATYKVNDELAALVNVEAGSEITYKHLISLLGFKMSVNVE GCHNMFITRDEAIRNVRGWVGFDVEATHACGTNIGTNLPFQVGFSTGADFVVTPEGLVDTSIGNNF EPVNSKAPPGEQFNHLRALFKSAKPWHVVRPRIVQMLADNLCNVSDCVVFVTWCHGLELTTLRYFV KIGKDQVCSCGSRATTFNSHTQAYACWKHCLGFDFVYNPLLVDIQQWGYSGNLQFNHDLHCNVHGH AHVASADAIMTRCLAINNAFCQDVNWDLTYPHIANEDEVNSSCRYLQRMYLNACVDALKVNVVYDI GNPKGIKCVRRGDLNFRFYDKNPIVPNVKQFEYDYNQHKDKFADGLCMFWNCNVDCYPDNSLVCRY DTRNLSVFNLPGCNGGSLYVNKHAFHTPKFDRTSFRNLKAMPFFFYDSSPCETIQLDGVAQDLVSL ATKDCITKCNICGAVCKKKAQMYADFVTSYNAAVTAGFTFWVTNNFNPYNLWKSFSALQ (nsp-15 amino acid sequence) SEQ ID NO: 8 SIDNIAYNMYKGGHYDAIAGEMPTIVTGDKVFVIDQGVEKAVFFNQTILPTSVAFELYAKRNIRTL PNNRILKGLGVDVTNGFVIWDYTNQTPLYRNTVKVCAYTDIEPNGLIVLYDDRYGDYQSFLAADNA VLVSTQCYKRYSYVEIPSNLLVQNGIPLKDGANLYVYKRVNGAFVTLPNTLNTQGRSYETFEPRSD VERDFLDMSEESFVEKYGKELGLQHILYGEVDKPQLGGLHTVIGMCRLLRANKLNAKSVTNSDSDV MQNYFVLADNGSYKQVCTVVDLLLDDFLELLRNILKEYGTNKSKVVTVSIDYHSINFMTWFEDGII KTCYPQLQ (nsp-16 amino acid sequence) SEQ ID NO: 9 SAWTCGYNMPELYKVQNCVMEPCNIPNYGVGIALPSGIMMNVAKYTQLCQYLSKTTMCVPHNMRVM HFGAGSDKGVAPGSTVLKQWLPEGTLLVDNDIVDYVSDAHVSVLSDCNKYKTEHKFDLVISDMYTD NDSKRKHEGVIANNGNDDVFIYLSSFLRNNLALGGSFAVKVTETSWHEVLYDIAQDCAWWTMFCTA VNASSSEAFLVGVNYLGASEKVIWSGKTLHANYIFWRNCNYLQTSAYSIFDVAKFDLRLKATPVVN LKTEQKTDLVFNLIKCGKLLVRDVGNTSFTSDSFVCTM
Reduced Pathogenicity
The live, attenuated coronavirus of the present invention comprises a variant replicase gene which causes the virus to have reduced pathogenicity compared to a coronavirus expressing the corresponding wild-type gene.
The term "attenuated" as used herein, refers to a virus that exhibits said reduced pathogenicity and may be classified as non-virulent. A live, attenuated virus is a weakened replicating virus still capable of stimulating an immune response and producing immunity but not causing the actual illness.
The term "pathogenicity" is used herein according to its normal meaning to refer to the potential of the virus to cause disease in a subject. Typically the pathogenicity of a coronavirus is determined by assaying disease associated symptoms, for example sneezing, snicking and reduction in tracheal ciliary activity.
The term "reduced pathogenicity" is used to describe that the level of pathogenicity of a coronavirus is decreased, lessened or diminished compared to a corresponding, wild-type coronavirus.
In one embodiment, the coronavirus of the present invention has a reduced pathogenicity compared to the parental M41-CK virus from which it was derived or a control coronavirus. The control coronavirus may be a coronavirus with a known pathogenicity, for example a coronavirus expressing the wild-type replicase protein.
The pathogenicity of a coronavirus may be assessed utilising methods well-known in the art. Typically, pathogenicity is assessed by assaying clinical symptoms in a subject challenged with the virus, for example a chicken.
As an illustration, the chicken may be challenged at 8-24 days old by nasal or ocular inoculation. Clinical symptoms, associated with IBV infection, may be assessed 3-10 days post-infection. Clinical symptoms commonly assessed to determine the pathogenicity of a coronavirus, for example an IBV, include gasping, coughing, sneezing, snicking, depression, ruffled feathers and loss of tracheal ciliary activity.
The variant replicase of the present invention, when expressed in a coronavirus, may cause a reduced level of clinical symptoms compared to a coronavirus expressing a wild-type replicase.
For example a coronavirus expressing the variant replicase may cause a number of snicks per bird per minute which is less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20% or less than 10% of the number of snicks caused by a virus expressing the wild type replicase.
A coronavirus expressing a variant replicase according to the present invention may cause wheezing in less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20% or less than 10% of the number of birds in a flock infected with the a virus expressing the wild type replicase.
A coronavirus expressing a variant replicase according to the present invention may result in tracheal ciliary activity which is at least 60%, at least 70%, at least 80%, at least 90% or at least 95% of the level of tracheal ciliary activity in uninfected birds.
A coronavirus expressing a variant replicase according to the present invention may cause clinical symptoms, as defined in Table 2, at a lower level than a coronavirus expressing the wild type replicase.
TABLE-US-00004 TABLE 2 IBV severity limits based on clinical signs: ##STR00001##
The variant replicase of the present invention, when expressed in a coronavirus, may cause the virus to replicate at non-pathogenic levels in ovo.
While developing vaccines to be administered in ovo to chicken embryos, attention must be paid to two points: the effect of maternal antibodies on the vaccines and the effect of the vaccines on the embryo. Maternal antibodies are known to interfere with active immunization. For example, vaccines with mild strains do not induce protective antibody levels when administered to broiler chickens with maternal antibodies as these strains are neutralized by the maternal antibody pool.
Thus a viral particle must be sufficiently efficient at replicating and propagating to ensure that it is not neutralized by the maternally-derived antibodies against the virus. Maternally-derived antibodies are a finite pool of effective antibodies, which decrease as the chicken ages, and neutralization of the virus in this manner does not equate to the establishment of long-term immunity for the embryo/chick. In order to develop long-term immunity against the virus, the embryo and hatched chicken must develop an appropriate protective immune response which is distinct to the effect of the maternally-derived antibodies.
To be useful for in ovo vaccination, the virus must also not replicate and propagate at a level which causes it to be pathogenic to the embryo.
Reduced pathogenicity in terms of the embryo may mean that the coronavirus causes less reduction in hatchability compared to a corresponding, wild-type control coronavirus. Thus the term "without being pathogenic to the embryo" in the context of the present invention may mean "without causing reduced hatchability" when compared to a control coronavirus.
A suitable variant replicase may be identified using methods which are known in the art. For example comparative challenge experiments following in ovo vaccination of embryos with or without maternally-derived antibodies may be performed (i.e. wherein the layer has or has not been vaccinated against IBV).
If the variant replicase enables the virus to propagate at a level which is too high, the embryo will not hatch or will not be viable following hatching (i.e. the virus is pathogenic to the embryo). A virus which is pathogenic to the embryo may kill the embryo.
If the variant replicase causes a reduction in viral replication and propagation which is too great, the virus will be neutralised by the maternally-derived antibodies. Subsequent challenge of the chick with IBV will therefore result in the development of clinical symptoms (for example wheezing, snicking, loss of ciliary activity) and the onset of disease in the challenged chick; as it will have failed to develop effective immunity against the virus.
Variant
As used herein, the term `variant` is synonymous with `mutant` and refers to a nucleic acid or amino acid sequence which differs in comparison to the corresponding wild-type sequence.
A variant/mutant sequence may arise naturally, or may be created artificially (for example by site-directed mutagenesis). The mutant may have at least 70, 80, 90, 95, 98 or 99% sequence identity with the corresponding portion of the wild type sequence. The mutant may have less than 20, 10, 5, 4, 3, 2 or 1 mutation(s) over the corresponding portion of the wild-type sequence.
The term "wild type" is used to mean a gene or protein having a nucleotide or amino acid sequence which is identical with the native gene or protein respectively (i.e. the viral gene or protein).
Identity comparisons can be conducted by eye, or more usually, with the aid of readily available sequence comparison programs. These commercially available computer programs can calculate % identity between two or more sequences. A suitable computer program for carrying out such an alignment is the GCG Wisconsin Bestfit package (University of Wisconsin, U.S.A.; Devereux et al., 1984, Nucleic Acids Research 12:387). Examples of other software that can perform sequence comparisons include, but are not limited to, the BLAST package (see Ausubel et al., 1999 ibid--Chapter 18), FASTA (Atschul et al., 1990, J. Mol. Biol., 403-410) and the GENEWORKS suite of comparison tools, ClustalX (see Larkin et al. (2007) Clustal W and Clustal X version 2.0. Bioinformatics, 23:2947-2948). Both BLAST and FASTA are available for offline and online searching (see Ausubel et al., 1999 ibid, pages 7-58 to 7-60). However, for some applications, it is preferred to use the GCG Bestf it program. A new tool, called BLAST 2 Sequences is also available for comparing protein and nucleotide sequence (see FEMS Microbiol Lett 1999 174(2): 247-50; FEMS Microbiol Lett 1999 177(1): 187-8 and
tatiana@ncbi.nlm.nih.gov).
The sequence may have one or more deletions, insertions or substitutions of amino acid residues which produce a silent change and result in a functionally equivalent molecule. Deliberate amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues as long as the activity is retained. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with uncharged polar head groups having similar hydrophilicity values include leucine, isoleucine, valine, glycine, alanine, asparagine, glutamine, serine, threonine, phenylalanine, and tyrosine.
Conservative substitutions may be made, for example according to the Table below. Amino acids in the same block in the second column and preferably in the same line in the third column may be substituted for each other:
TABLE-US-00005 ALIPHATIC Non-polar G A P I L V Polar- uncharged C S T M N Q Polar- charged D E K R AROMATIC H F W Y
The coronavirus of the present invention may comprise a variant replicase gene which encodes a protein which comprises a mutation compared to any one of SEQ ID NO: 6, 7, 8 or 9 which, when expressed in a coronavirus, causes the virus to have reduced pathogenicity compared to a coronavirus expressing the corresponding wild-type replicase.
The variant replicase gene may encode a protein which comprises at least one or more amino acid mutations in any combination of nsp-10, nsp-14, nsp-15 and nsp-16.
The variant replicase gene of the coronavirus of the present invention may encode a protein comprising a mutation as defined in the M41 mod sequences presented in FIG. 10.
The variant replicase gene of the coronavirus of the present invention may encode a protein which comprises one or more amino acid mutations selected from the list of: Pro to Leu at position 85 of SEQ ID NO: 6, Val to Leu at position 393 of SEQ ID NO: 7; Leu to Ile at position 183 of SEQ ID NO: 8; Val to Ile at position 209 of SEQ ID NO: 9.
The variant replicase gene of the coronavirus of the present invention may encode a protein which does not comprise a mutation in nsp-2, nsp-3, nsp-6 or nsp-13.
The variant replicase gene of the coronavirus of the present invention may encode a protein which does not comprise a mutation in nsp10 which corresponds to the threonine to isoleucine mutation caused by a mutation at nucleotide position 12,008 in the gene reported by Ammayappan et al. (Arch Virol (2009) 154:495-499).
Ammayappan et al (as above) reports the identification of sequence changes responsible for the attenuation of IBV strain Arkansas DPI. The study identified 17 amino acid changes in a variety of IBV proteins following multiple passages, approx. 100, of the virus in embryonated eggs. It was not investigated whether the attenuated virus (Ark DPI 101) is capable of replicating in the presence of maternally-derived antibodies against the virus in ovo, without being pathogenic to the embryo. Given that this virus was produced by multiple passage in SPF embryonated eggs, similar methodology for classical IBV vaccines, it is likely that this virus is pathogenic for embryos. The virus may also be sensitive to maternally-derived antibodies if the hens were vaccinated with a similar serotype.
The variant replicase gene of the coronavirus of the present invention may encode a protein which comprises any combination of one or more amino acid mutations provided in the list above.
The variant replicase gene may encode a protein which comprises the amino acid mutation Pro to Leu at position 85 of SEQ ID NO: 6.
The variant replicase gene may encode a protein which comprises the amino acid mutation Val to Leu at position 393 of SEQ ID NO: 7.
The variant replicase gene may encode a protein which comprises the amino acid mutation Leu to Ile at position 183 of SEQ ID NO: 8.
The variant replicase gene may encode a protein which comprises the amino acid mutation Val to Ile at position 209 of SEQ ID NO: 9.
The variant replicase gene may encode a protein which comprises the amino acid mutations Pro to Leu at position 85 of SEQ ID NO: 6, and Val to Leu at position 393 of SEQ ID NO: 7.
The variant replicase gene may encode a protein which comprises the amino acid mutations Pro to Leu at position 85 of SEQ ID NO: 6 Leu to Ile at position 183 of SEQ ID NO: 8.
The variant replicase gene may encode a protein which comprises the amino acid mutations Pro to Leu at position 85 of SEQ ID NO: 6 and Val to Ile at position 209 of SEQ ID NO: 9.
The variant replicase gene may encode a protein which comprises the amino acid mutations Val to Leu at position 393 of SEQ ID NO: 7 and Leu to Ile at position 183 of SEQ ID NO: 8.
The variant replicase gene may encode a protein which comprises the amino acid mutations Val to Leu at position 393 of SEQ ID NO: 7 and Val to Ile at position 209 of SEQ ID NO: 9.
The variant replicase gene may encode a protein which comprises the amino acid mutations Leu to Ile at position 183 of SEQ ID NO: 8 and Val to Ile at position 209 of SEQ ID NO: 9.
The variant replicase gene may encode a protein which comprises the amino acid mutations Pro to Leu at position 85 of SEQ ID NO: 6, Val to Leu at position 393 of SEQ ID NO: 7 and Leu to Ile at position 183 of SEQ ID NO: 8.
The variant replicase gene may encode a protein which comprises the amino acid mutations Pro to Leu at position 85 of SEQ ID NO: 6 Leu to Ile at position 183 of SEQ ID NO: 8 and Val to Ile at position 209 of SEQ ID NO: 9.
The variant replicase gene may encode a protein which comprises the amino acid mutations Pro to Leu at position 85 of SEQ ID NO: 6, Val to Leu at position 393 of SEQ ID NO: 7 and Val to Ile at position 209 of SEQ ID NO: 9.
The variant replicase gene may encode a protein which comprises the amino acid mutations Val to Leu at position 393 of SEQ ID NO: 7, Leu to Ile at position 183 of SEQ ID NO: 8 and Val to Ile at position 209 of SEQ ID NO: 9.
The variant replicase gene may encode a protein which comprises the amino acid mutations Pro to Leu at position 85 of SEQ ID NO: 6, Val to Leu at position 393 of SEQ ID NO: 7, Leu to Ile at position 183 of SEQ ID NO: 8 and Val to Ile at position 209 of SEQ ID NO: 9.
The variant replicase gene may also be defined at the nucleotide level.
For example the nucleotide sequence of the variant replicase gene of the coronavirus of the present invention may comprise one or more nucleotide substitutions within the regions selected from the list of: 11884-12318, 16938-18500, 18501-19514 and 19515-20423 of SEQ ID NO:1.
For example the nucleotide sequence of the variant replicase gene of the coronavirus of the present invention may comprise one or more nucleotide substitutions selected from the list of: C to Tat nucleotide position 12137; G to C at nucleotide position 18114; T to A at nucleotide position 19047; and G to A at nucleotide position 20139; compared to the sequence shown as SEQ ID NO: 1.
As used herein, the term "substitution" is synonymous with the term mutation and means that the nucleotide at the identified position differs to that of the wild-type nucleotide sequence.
The nucleotide sequence may comprise any combination of the nucleotide substitutions selected from the list of: C to Tat nucleotide position 12137; G to Cat nucleotide position 18114; T to A at nucleotide position 19047; and G to A at nucleotide position 20139; compared to the sequence shown as SEQ ID NO: 1.
The nucleotide sequence may comprise the substitution C12137T.
The nucleotide sequence may comprise substitution G18114C.
The nucleotide sequence may comprise the substitution T19047A.
The nucleotide sequence may comprise the substitution G20139A.
The nucleotide sequence may comprise the substitutions C12137T and G18114C.
The nucleotide sequence may comprise the substitutions C12137T and T19047A.
The nucleotide sequence may comprise the substitutions C12137T and G20139A.
The nucleotide sequence may comprise the substitutions G18114C and T19047A.
The nucleotide sequence may comprise the substitutions G18114C and G20139A.
The nucleotide sequence may comprise the substitutions T19047A and G20139A.
The nucleotide sequence may comprise the substitutions C12137T, G18114C and T19047A.
The nucleotide sequence may comprise the substitutions C12137T, T19047A and G20139A.
The nucleotide sequence may comprise the substitutions C12137T, G18114C and G20139A.
The nucleotide sequence may comprise the substitutions G18114C, T19047A and G20139A.
The nucleotide sequence may comprise the substitutions C12137T, G18114C, T19047A and G20139A.
The nucleotide sequence may not comprise a substitution which corresponds to the C12008T substitution reported by Ammayappan et al. (as above).
The nucleotide sequence may be natural, synthetic or recombinant. It may be double or single stranded, it may be DNA or RNA or combinations thereof. It may, for example, be cDNA, PCR product, genomic sequence or mRNA.
The nucleotide sequence may be codon optimised for production in the host/host cell of choice.
It may be isolated, or as part of a plasmid, virus or host cell.
Plasmid
A plasmid is an extra-chromosomal DNA molecule separate from the chromosomal DNA which is capable of replicating independently of the chromosomal DNA. They are usually circular and double-stranded.
Plasmids, or vectors (as they are sometimes known), may be used to express a protein in a host cell. For example a bacterial host cell may be transfected with a plasmid capable of encoding a particular protein, in order to express that protein. The term also includes yeast artificial chromosomes and bacterial artificial chromosomes which are capable of accommodating longer portions of DNA.
The plasmid of the present invention comprises a nucleotide sequence capable of encoding a defined region of the replicase protein. It may also comprise one or more additional coronavirus nucleotide sequence(s), or nucleotide sequence(s) capable of encoding one or more other coronavirus proteins such as the S gene and/or gene 3.
The plasmid may also comprise a resistance marker, such as the guanine xanthine phosphoribosyltransferase gene (gpt) from Escherichia coli, which confers resistance to mycophenolic acid (MPA) in the presence of xanthine and hypoxanthine and is controlled by the vaccinia virus P7.5 early/late promoter.
Recombinant Vaccinia Virus
The present invention also relates to a recombinant vaccinia virus (rVV) comprising a variant replicase gene as defined herein.
The recombinant vaccinia virus (rVV) may be made using a vaccinia-virus based reverse genetics system.
In this respect, the present invention also provides a method for making a viral particle by: (i) transfecting a plasmid as described in the previous section into a host cell; (ii) infecting the host cell with a recombining virus comprising the genome of a coronavirus strain with a replicase gene; (iii) allowing homologous recombination to occur between the replicase gene sequences in the plasmid and the corresponding sequences in the recombining virus genome to produce a modified replicase gene; (iv) selecting for recombining virus comprising the modified replicase gene.
The term `modified replicase gene` refers to a replicase gene which comprises a variant replicase gene as described in connection with the first aspect of the present invention. Specifically, the term refers to a gene which is derived from a wild-type replicase gene but comprises a nucleotide sequence which causes it to encode a variant replicase protein as defined herein.
The recombination may involve all or part of the replicase gene. For example the recombination may involve a nucleotide sequence encoding for any combination of nsp-10, nsp-14, nsp-15 and/or nsp-16. The recombination may involve a nucleotide sequence which encodes for an amino acid mutation or comprises a nucleotide substitution as defined above.
The genome of the coronavirus strain may lack the part of the replicase protein corresponding to the part provided by the plasmid, so that a modified protein is formed through insertion of the nucleotide sequence provided by the plasmid.
The recombining virus is one suitable to allow homologous recombination between its genome and the plasmid. The vaccinia virus is particularly suitable as homologous recombination is routinely used to insert and delete sequences for the vaccinia virus genome.
The above method optionally includes the step: (v) recovery of recombinant coronavirus comprising the modified replicase gene from the DNA from the recombining virus from step (iv).
Methods for recovering recombinant coronavirus, such as recombinant IBV, are known in the art (See Britton et al (2005) see page 24; and PCT/GB2010/001293).
For example, the DNA from the recombining virus from step (iv) may be inserted into a plasmid and used to transfect cells which express cytoplasmic T7 RNA polymerase. The cells may, for example be pre-infected with a fowlpox virus expressing T7 RNA polymerase. Recombinant coronavirus may then be isolated, for example, from the growth medium.
When the plasmid is inserted into the vaccinia virus genome, an unstable intermediate is formed. Recombinants comprising the plasmid may be selected for e.g. using a resistance marker on the plasmid.
Positive recombinants may then be verified to contain the modified replicase gene by, for example, PCR and sequencing.
Large stocks of the recombining virus including the modified replicase gene (e.g. recombinant vaccinia virus, (rVV) may be grown up and the DNA extracted in order to carry out step (v)).
Suitable reverse genetics systems are known in the art (Casais et al (2001) J. Virol 75:12359-12369; Casais et al (2003) J. Virol. 77:9084-9089; Britton et al (2005) J. Virological Methods 123:203-211; Armesto et al (2008) Methods in Molecular Biology 454:255-273).
Cell
The coronavirus may be used to infect a cell.
Coronavirus particles may be harvested, for example from the supernatant, by methods known in the art, and optionally purified.
The cell may be used to produce the coronavirus particle.
Thus the present invention also provides a method for producing a coronavirus which comprises the following steps:
(i) infection of a cell with a coronavirus according to the invention;
(ii) allowing the virus to replicate in the cell; and
(iii) harvesting the progeny virus.
The present invention also provides a cell capable of producing a coronavirus according to the invention using a reverse genetics system. For example, the cell may comprise a recombining virus genome comprising a nucleotide sequence capable of encoding the replicase gene of the present invention.
The cell may be able to produce recombinant recombining virus (e.g. vaccinia virus) containing the replicase gene.
Alternatively the cell may be capable of producing recombinant coronavirus by a reverse genetics system. The cell may express or be induced to express T7 polymerase in order to rescue the recombinant viral particle.
Vaccine
The coronavirus may be used to produce a vaccine. The vaccine may by a live attenuated form of the coronavirus of the present invention and may further comprise a pharmaceutically acceptable carrier. As defined herein, "pharmaceutically acceptable carriers" suitable for use in the invention are well known to those of skill in the art. Such carriers include, without limitation, water, saline, buffered saline, phosphate buffer, alcohol/aqueous solutions, emulsions or suspensions. Other conventionally employed diluents and excipients may be added in accordance with conventional techniques. Such carriers can include ethanol, polyols, and suitable mixtures thereof, vegetable oils, and injectable organic esters. Buffers and pH adjusting agents may also be employed. Buffers include, without limitation, salts prepared from an organic acid or base. Representative buffers include, without limitation, organic acid salts, such as salts of citric acid, e.g., citrates, ascorbic acid, gluconic acid, histidine-Hel, carbonic acid, tartaric acid, succinic acid, acetic acid, or phthalic acid, Iris, trimethanmine hydrochloride, or phosphate buffers. Parenteral carriers can include sodium chloride solution, Ringer's dextrose, dextrose, trehalose, sucrose, and sodium chloride, lactated Ringer's or fixed oils. Intravenous carriers can include fluid and nutrient replenishers, electrolyte replenishers, such as those based on Ringer's dextrose and the like. Preservatives and other additives such as, for example, antimicrobials, antioxidants, chelating agents (e.g., EDTA), inert gases and the like may also be provided in the pharmaceutical carriers. The present invention is not limited by the selection of the carrier. The preparation of these pharmaceutically acceptable compositions, from the above-described components, having appropriate pH isotonicity, stability and other conventional characteristics is within the skill of the art. See, e.g., texts such as Remington: The Science and Practice of Pharmacy, 20th ed, Lippincott Williams & Wilkins, pub!., 2000; and The Handbook of Pharmaceutical Excipients, 4.sup.th edit., eds. R. C. Rowe et al, APhA Publications, 2003.
The vaccine of the invention will be administered in a "therapeutically effective amount", which refers to an amount of an active ingredient, e.g., an agent according to the invention, sufficient to effect beneficial or desired results when administered to a subject or patient. An effective amount can be administered in one or more administrations, applications or dosages. A therapeutically effective amount of a composition according to the invention may be readily determined by one of ordinary skill in the art. In the context of this invention, a "therapeutically effective amount" is one that produces an objectively measured change in one or more parameters associated Infectious Bronchitis condition sufficient to effect beneficial or desired results. An effective amount can be administered in one or more administrations. For purposes of this invention, an effective amount of drug, compound, or pharmaceutical composition is an amount sufficient to reduce the incidence of Infectious Bronchitis. As used herein, the term "therapeutic" encompasses the full spectrum of treatments for a disease, condition or disorder. A "therapeutic" agent of the invention may act in a manner that is prophylactic or preventive, including those that incorporate procedures designed to target animals that can be identified as being at risk (pharmacogenetics); or in a manner that is ameliorative or curative in nature; or may act to slow the rate or extent of the progression of at least one symptom of a disease or disorder being treated.
The present invention also relates to a method for producing such a vaccine which comprises the step of infecting cells, for example Vero cells, with a viral particle comprising a replicase protein as defined in connection with the first aspect of the invention.
Vaccination Method
The coronavirus of the present invention may be used to treat and/or prevent a disease.
To "treat" means to administer the vaccine to a subject having an existing disease in order to lessen, reduce or improve at least one symptom associated with the disease and/or to slow down, reduce or block the progression of the disease.
To "prevent" means to administer the vaccine to a subject who has not yet contracted the disease and/or who is not showing any symptoms of the disease to prevent or impair the cause of the disease (e.g. infection) or to reduce or prevent development of at least one symptom associated with the disease.
The disease may be any disease caused by a coronavirus, such as a respiratory disease and and/or gastroenteritis in humans and hepatitis, gastroenteritis, encephalitis, or a respiratory disease in other animals.
The disease may be infectious bronchitis (IB); Porcine epidemic diarrhoea; Transmissible gastroenteritis; Mouse hepatitis virus; Porcine haemagglutinating encephalomyelitis; Severe acute respiratory syndrome (SARS); or Bluecomb disease.
The disease may be infectious bronchitis.
The vaccine may be administered to hatched chicks or chickens, for example by eye drop or intranasal administration. Although accurate, these methods can be expensive e.g. for large broiler flocks. Alternatives include spray inoculation of administration to drinking water but it can be difficult to ensure uniform vaccine application using such methods.
The vaccine may be provided in a form suitable for its administration, such as an eye-dropper for intra-ocular use.
The vaccine may be administered by in ovo inoculation, for example by injection of embryonated eggs. In ovo vaccination has the advantage that it provides an early stage resistance to the disease. It also facilitates the administration of a uniform dose per subject, unlike spray inoculation and administration via drinking water.
The vaccine may be administered to any suitable compartment of the egg, including allantoic fluid, yolk sac, amnion, air cell or embryo. It may be administered below the shell (aircell) membrane and chorioallantoic membrane.
Usually the vaccine is injected into embryonated eggs during late stages of embryonic development, generally during the final quarter of the incubation period, such as 3-4 days prior to hatch. In chickens, the vaccine may be administered between day 15-19 of the 21-day incubation period, for example at day 17 or 18.
The process can be automated using a robotic injection process, such as those described in WO 2004/078203.
The vaccine may be administered together with one or more other vaccines, for example, vaccines for other diseases, such as Newcastle disease virus (NDV). The present invention also provides a vaccine composition comprising a vaccine according to the invention together with one or more other vaccine(s). The present invention also provides a kit comprising a vaccine according to the invention together with one or more other vaccine(s) for separate, sequential or simultaneous administration.
The vaccine or vaccine composition of the invention may be used to treat a human, animal or avian subject. For example, the subject may be a chick, chicken or mouse (such as a laboratory mouse, e.g. transgenic mouse).
Typically, a physician or veterinarian will determine the actual dosage which will be most suitable for an individual subject or group of subjects and it will vary with the age, weight and response of the particular subject(s).
The composition may optionally comprise a pharmaceutically acceptable carrier, diluent, excipient or adjuvant. The choice of pharmaceutical carrier, excipient or diluent can be selected with regard to the intended route of administration and standard pharmaceutical practice. The pharmaceutical compositions may comprise as (or in addition to) the carrier, excipient or diluent, any suitable binder(s), lubricant(s), suspending agent(s), coating agent(s), solubilising agent(s), and other carrier agents that may aid or increase the delivery or immunogenicity of the virus.
The invention will now be further described by way of Examples, which are meant to serve to assist one of ordinary skill in the art in carrying out the invention and are not intended in any way to limit the scope of the invention.
EXAMPLES
Example 1--Generation of an IBV Reverse Genetics System Based on M41-CK
A M41-CK full-length cDNA was produced by replacement of the Beaudette cDNA in the Vaccinia virus reverse genetics system previously described in PCT/GB2010/001293 (herein incorporated by reference) with synthetic cDNA derived from the M41 consensus sequence.
The IBV cDNA within recombinant Vaccinia virus (rVV) rVV-BeauR-Rep-M41 structure described in Armesto, Cavanagh and Britton (2009). PLoS ONE 4(10): e7384. doi:10.1371/journal.pone.0007384, which consisted of the replicase derived from IBV Beaudette strain and the structural and accessory genes and 3' UTR from IBV M41-CK, was further modified by replacement of the Beaudette 5' UTR-Nsp2-Nsp3 sequence with the corresponding sequence from IBV M41-CK. The resulting IBV cDNA consisted of 5' UTR-Nsp2-Nsp3 from M41, Nsp4-Nsp16 from Beaudette and the structural and accessory genes and 3' UTR from M41. This cDNA was further modified by the deletion of the Beaudette Nsp4-Nsp16 sequence. The resulting cDNA, lacking Nsp4-16, was modified in four further steps in which the deleted Nsps were sequentially replaced with the corresponding sequences from M41-CK, the replacement cDNAs represented M41-CK Nsp4-8, Nsp9-12, Nsp12-14 and finally Nsp15-16. Each replacement cDNA contained approx. 500 nucleotides at the 5' end corresponding to the 3' most M41 sequence previously inserted and approx. 500 nucleotides at the 3' end corresponding to the M41 S gene sequence. This allowed insertion of the M41 cDNA sequence by homologous recombination and sequential addition of contiguous M41 replicase gene sequence. The synthetic cDNAs containing the M41-derived Nsp sequences were added by homologous recombination utilising the inventor's previous described transient dominant selection (IDS) system (see PCT/GB2010/001293). The M41-derived cDNAs containing sequence corresponding to the M41 Nsps-10, -14, -15 and -16 contained the modified amino acids at positions 85, 393, 183 and 209, respectively, as indicated in FIG. 10.
A full-length cDNA representing the genome of M41-CK was generated in Vaccinia virus representing the synthetic sequences. Two rIBVs, M41-R-6 and M41-R-12, were rescued and shown to grow in a similar manner as M41-CK (FIG. 1).
Example 2--Determining the Pathogenicity of Rescued M41 Viruses
The viruses rescued in Example 1 were used to infect 8-day-old specific pathogen free (SPF) chicks by ocular and nasal inoculation to test them for pathogenicity, as observed by clinical signs on a daily basis 3-7 days post-infection and for ciliary activity days 4 and 6 post-infection. Loss of ciliary activity is a well-established method for determining the pathogenicity of IBV. The two M41-R viruses were found to be apathogenic when compared to M41-CK though they did show some clinical signs in comparison to uninfected control chicks (FIG. 2) and some but inconsistent loss in ciliary activity (FIG. 3).
Thus, the M41-R molecular clones of M41-CK were not pathogenic when compared to the parental virus M41-CK.
The inventors identified several nucleotide differences in the M41-R compared to the M41-CK sequences. The majority of these were synonymous mutations, as the nucleotide change did not affect the amino acid sequence of the protein associated with the sequence. However, four non-synonymous mutations were identified in the IBV replicase gene specific to Nsp-10, Nsp-14, Nsp-15 and Nsp-16 components of the replicase gene, these mutations resulted in amino acid changes (Table 3).
TABLE-US-00006 TABLE 3 Non-Synonymous mutations identified in the Nsps of M41-R full-length genome Region of Nucleotide Nucleotide Replicase position Mutation Amino Acid Change Nsp10 12137 C.fwdarw.T Pro.fwdarw.Leu Nsp14 18114 G.fwdarw.C Val.fwdarw.Leu Nsp15 19047 T.fwdarw.A Leu.fwdarw.Ile Nsp16 20139 G.fwdarw.A Val.fwdarw.Ile
Example 3--Repair of M41-R rIBVs
In order to determine whether the identified mutations were responsible for the loss of pathogenicity associated with M41-R, the Nsp10 mutation was repaired and the mutations in Nsp-14, -15 & -16 were repaired and shown to grow in a similar manner as M41-CK (FIG. 9). The inventors thus generated the rIBVs, M41R-nsp10rep and M41R-nsp14, 15, 16rep, using synthetic cDNAs containing the correct nucleotides utilising the inventor's previous described (TDS) system (see PCT/GB2010/001293).
The rIBVs were assessed for pathogenicity in chicks as described previously. Both rIBVs showed increased pathogenicity when compared to M41-R but not to the level observed with M41-CK (FIGS. 4 and 5). M41R-nsp14, 15, 16rep gave more clinical signs and more reduction in ciliary activity than M41R-nsp10rep, overall these results indicated that the changes associated with the four Nsps appear to affect pathogenicity.
To determine the roles of the Nsps in pathogenicity the full-length cDNA corresponding to M41R-nsp10rep was used to repair the mutations in Nsps14, 15 & 16 using a synthetic cDNA containing the correct nucleotides utilising the TDS system.
The following rIBVs were produced:
M41R-nsp10, 15rep--M41-R with the mutations in Nsp-10 and Nsp-15 repaired
M41R-nsp10, 14, 15rep--M41-R with mutations in Nsp-10, -14 and -15 repaired
M41R-nsp10, 14, 16rep--M41-R with mutations in Nsp-10, -14 and -16 repaired
M41R-nsp10, 15, 16rep--M41-R with mutations in Nsp-10, -15 and -16 repaired
M41-K--All four mutations, Nsp-10, -14, -15 & -16 repaired in M41-R
The rIBVs were shown to grow in a similar manner as M41-CK (FIG. 9) and assessed for pathogenicity as described previously. M41-K (in which all four mutations had been repaired) resulted in clinical signs and 100% loss of ciliary activity (complete ciliostasis) by 4 days post-infection (FIGS. 6, 7 & 8). The other rIBVs demonstrated varying levels of pathogenicity, apart from M41R-nsp10, 15, 16rep, which was essentially apathogenic. These results confirmed that repair of all four Nsps restored pathogenicity to M41-R; again supporting the previous evidence that the mutations described in the four Nsps are implicated in attenuating M41-CK.
The inventors also generated rIBV M41R-nsp 10, 14 rep (nsp 10 and 14 are repaired, nsp 15 and 16 contain mutations) and rIBV M41R-nsp 10, 16 rep (nsp 10 and 16 are repaired, nsp 14 and 15 contain mutations) and assessed the pathogenicity of these viruses.
rIBV M41R-nsp 10, 14 rep less pathogenic than M41-K but caused around 50% ciliostasis on days 4-6 post-infection. rIBV M41R-nsp 10, 16 rep was almost apathogenic and caused no ciliostasis (see FIG. 11a-c).
Thus the genome associated with M41-R is a potential backbone genome for a rationally attenuated IBV.
Example 4--Vaccination/Challenge Study with M41-R
Candidate vaccine viruses were tested in studies in which fertilized chicken eggs were vaccinated in ovo at 18 days embryonation and in which the hatchability of the inoculated eggs was determined. The clinical health of the chickens was investigated and the chickens were challenged at 21 days of age with a virulent IB M41 challenge virus at 10.sup.3.65 EID.sub.50 per dose.
Clinical signs were investigated after challenge protection by the vaccine and a ciliostasis test was performed at 5 days after challenge to investigate the effect of the challenge viruses on movement of the cilia and protection by the vaccine against ciliostasis (inhibition of cilia movement).
In Ovo Vaccination in Commercial Broiler Eggs
The design of the experiment is given in Table 4 and the clinical results are given in Table 5. Hatchability of the eggs inoculated with IB M41-R was good and chickens were healthy. IB M41-R protected against clinical signs after challenge in the broilers (placebo: 19/19 affected, 1B M41-R: 3/18 affected and 1 dead). The results of the ciliostasis test are given in Table 6. IB M41-R generated protection against ciliostasis.
TABLE-US-00007 TABLE 4 Design of a hatchability, safety, efficacy study in commercial eggs EID.sub.50.sup.1 Route Day(s) Day(s) End Nr. of Treatment per of of of of eggs per Treatment Description dose Admin Admin Challenge.sup.2 Study treatment T01 None NA NA NA NA NA 30 T02 IB M41-R 10.sup.4 In ovo 18 days At 21 days At 26 30 NTX Saline NA In ovo embryo- of age, 20 days 30 nation chickens of age per group .sup.1Dose volume 0.1 ml, NA, not applicable. .sup.210.sup.3.65 EID.sub.50 per dose.
TABLE-US-00008 TABLE 5 Hatch percentages and clinical data before and after challenge in commercial chickens, for design see Table 1. Before After challenge challenge Hatch/ Vital/ Deaths/ Symptoms/ Deaths/ Symptoms/ Treatment total total total total total total None 28/30 Euthanized directly after hatch for blood collection IB M41-R 28/30 28/28 1/20 0/19 1/19 3/18.sup.1, 7 Saline 29/30 29/29 1/20 0/19 0/19 19/19.sup.1, 2, 3, 4, 5, 6, 7 .sup.1Disturbed respiratory system .sup.2Whizzing .sup.3Change of voice .sup.4Breathing difficult .sup.5Swollen intra-orbital sinuses .sup.6Uneven growth .sup.7Weak
TABLE-US-00009 TABLE 6 Results of the ciliostasis test after challenge, for design see Table 1. Treatment Protected/total Percentage protection Saline 0/19 0% IB M41R 5/18 28%
In Ovo Vaccination in Specific Pathogen-Free (SPF) Eggs
The design of the study in SPF eggs is given in Table 7 and is similar with the design of the studies with commercial broilers, but the vaccination dose for 1B M41-R was higher, (10.sup.5 EID.sub.50 per dose).
The results (Table 8) show that the hatch percentage for IB M41-R hatch was low, and 19 of 40 hatched and the chicks were weak. Eight chicks died. The remaining 11 chickens were challenged and 11 of the chicks hatched from the eggs which had been inoculated with saline were challenged.
In the ciliostasis test after challenge it appeared that all chickens vaccinated in ovo with IB M41-R were protected, whereas none of the controls was protected, see Table 9.
TABLE-US-00010 TABLE 7 Design of a hatchability, safety, efficacy study in SPF eggs EID.sub.50.sup.1 Route Day Day End Nr. of Treatment per of of of of eggs per Treatment Description dose Admin Admin Challenge.sup.2 Study treatment T01 IB M41-R 10.sup.5 In ovo 18 days At 21 days At 26 40 embryo- of age days T04 Saline NA In ovo nation of age 40 NTX NA NA NA NA 10 .sup.1Dose volume 0.1 ml, NA, not applicable. .sup.2Challenge dose 10.sup.3.3 EID.sub.50 in 0.2 ml.
TABLE-US-00011 TABLE 8 Hatch percentages and clinical data before and after challenge in SPF chickens, for design see Table 7. Before After challenge challenge Hatch/ Vital/ Deaths/ Symptoms/ Deaths/ Symptoms/ Treatment total total total total total total IB M41-R 19/40 11/40 8/40 weak 0 0 Saline 30/40 30/40 0 -- 0 0 NA 9/10 9/10 0 -- -- --
TABLE-US-00012 TABLE 9 Results of the ciliostasis test after challenge, for design see Table 7. Treatment Protected/total Percentage protection Saline 0/11 0% IB M41R 11/11 100%
In conclusion, IB M41-R was safe in commercial eggs, generated protection against clinical signs and to an extent against ciliostasis.
In SPF eggs vaccinated with IB M41 R a relatively low number of chickens hatched. This may be due to the 10.sup.5 EID.sub.50 per egg of 1B M41-R used. This was 10-fold higher than the dose used in earlier studies in which there was a higher level of hatchability. The lower hatch percentages may also be caused by a particularly high susceptibility of the batch of SPF eggs for viruses, as in other studies the level of embryo mortality was also higher that had previously been observed.
After challenge all surviving chickens after hatch were completely protected against ciliostasis. It is concluded that IB M41-R has great potential as vaccine to be administered in ovo.
All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described methods and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in molecular biology, virology or related fields are intended to be within the scope of the following claims.
SEQUENCE LISTINGS
1
13127500DNAInfectious 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 275002435DNAInfectious bronchitis virus 2tctaaaggtc atgagacaga ggaagtggat gctgtaggca ttctctcact ttgttctttt 60gcagtagatc ctgcggatac atattgtaaa tatgtggcag caggtaatca acctttaggt 120aactgtgtta aaatgttgac agtacataat ggtagtggtt ttgcaataac atcaaagcca 180agtccaactc cggatcagga ttcttatgga ggagcttctg tgtgtcttta ttgtagagca 240catatagcac accttggcgg agcaggaaat ttagatggac gctgtcaatt taaaggttct 300tttgtgcaaa tacctactac ggagaaagat cctgttggat tctgtctacg taacaaggtt 360tgcactgttt gtcagtgttg gattggttat ggatgtcagt gtgattcact tagacaacct 420aaaccttctg ttcag 43531563DNAInfectious bronchitis virus 3ggtacaggct tgtttaaaat ttgcaacaaa gagtttagtg gtgttcaccc agcttatgca 60gtcacaacta aggctcttgc tgcaacttat aaagttaatg atgaacttgc tgcacttgtt 120aacgtggaag ctggttcaga aataacatat aaacatctta tttctttgtt agggtttaag 180atgagtgtta atgttgaagg ctgccacaac atgtttataa cacgtgatga ggctatccgc 240aacgtaagag gttgggtagg ttttgatgta gaagcaacac atgcttgcgg tactaacatt 300ggtactaacc tgcctttcca agtaggtttc tctactggtg cagactttgt agttacgcct 360gagggacttg tagatacttc aataggcaat aattttgagc ctgtgaattc taaagcacct 420ccaggtgaac aatttaatca cttgagagcg ttattcaaaa gtgctaaacc ttggcatgtt 480gtaaggccaa ggattgtgca aatgttagcg gataacctgt gcaacgtttc agattgtgta 540gtgtttgtca cgtggtgtca tggcctagaa ctaaccactt tgcgctattt tgttaaaata 600ggcaaggacc aagtttgttc ttgcggttct agagcaacaa cttttaattc tcatactcag 660gcttatgctt gttggaagca ttgcttgggt tttgattttg tttataatcc actcttagtg 720gatattcaac agtggggtta ttctggtaac ctacaattta accatgattt gcattgtaat 780gtgcatggac acgcacatgt agcttctgcg gatgctatta tgacgcgttg tcttgcaatt 840aataatgcat tttgtcaaga tgtcaactgg gatttaactt accctcatat agcaaatgag 900gatgaagtca attctagctg tagatattta caacgcatgt atcttaatgc atgtgttgat 960gctcttaaag ttaacgttgt ctatgatata ggcaacccta aaggtattaa atgtgttaga 1020cgtggagact taaattttag attctatgat aagaatccaa tagtacccaa tgtcaagcag 1080tttgagtatg actataatca gcacaaagat aagtttgctg atggtctttg tatgttttgg 1140aattgtaatg tggattgtta tcccgacaat tccttacttt gtaggtacga cacacgaaat 1200ttgagtgtgt ttaacctacc tggttgtaat ggtggtagct tgtatgttaa caagcatgca 1260ttccacacac ctaaatttga tcgcactagc tttcgtaatt tgaaagctat gccattcttt 1320ttctatgact catcgccttg cgagaccatt caattggatg gagttgcgca agaccttgtg 1380tcattagcta cgaaagattg tatcacaaaa tgcaacatag gcggtgctgt ttgtaaaaag 1440cacgcacaaa tgtatgcaga ttttgtgact tcttataatg cagctgttac tgctggtttt 1500actttttggg ttactaataa ttttaaccca tataatttgt ggaaaagttt ttcagctctc 1560cag 156341014DNAInfectious bronchitis virus 4tctatcgaca atattgctta taatatgtat aagggtggtc attatgatgc tattgcagga 60gaaatgccca ctatcgtaac tggagataaa gtttttgtta tagatcaagg cgtagaaaaa 120gcagtttttt ttaatcaaac aattctgcct acatctgtag cgtttgagct gtatgcgaag 180agaaatattc gcacactgcc aaacaaccgt attttgaaag gtttgggtgt agatgtgact 240aatggatttg taatttggga ttacacgaac caaacaccac tataccgtaa tactgttaag 300gtatgtgcat atacagacat agaaccaaat ggcctaatag tgctgtatga tgatagatat
360ggtgattacc agtcttttct agctgctgat aatgctgttt tagtttctac acagtgttac 420aagcggtatt cgtatgtaga aataccgtca aacctgcttg ttcagaacgg tattccgtta 480aaagatggag cgaacctgta tgtttataag cgtgttaatg gtgcgtttgt tacgctacct 540aacacaataa acacacaggg tcgaagttat gaaacttttg aacctcgtag tgatgttgag 600cgtgattttc tcgacatgtc tgaggagagt tttgtagaaa agtatggtaa agaattaggt 660ctacagcaca tactgtatgg tgaagttgat aagccccaat taggtggttt ccacactgtt 720ataggtatgt gcagactttt acgtgcgaat aagttgaacg caaagtctgt tactaattct 780gattctgatg tcatgcaaaa ttattttgta ttggcagaca atggttccta caagcaagtg 840tgtactgttg tggatttgct gcttgatgat ttcttagaac ttcttaggaa catactgaaa 900gagtatggta ctaataagtc taaagttgta acagtgtcaa ttgattacca tagcataaat 960tttatgactt ggtttgaaga tggcattatt aaaacatgtt atccacagct tcaa 10145909DNAInfectious bronchitis virus 5tcagcatgga cgtgtggtta taatatgcct gaactttata aagttcagaa ttgtgttatg 60gaaccttgca acattcctaa ttatggtgtt ggaatagcgt tgccaagtgg tattatgatg 120aatgtggcaa agtatacaca actctgtcaa tacctttcga aaacaacaat gtgtgtaccg 180cataatatgc gagtaatgca ttttggagct ggaagtgaca aaggagtggt gccaggtagt 240actgttctta aacaatggct cccagaaggg acactccttg tcgataatga tattgtagac 300tatgtgtctg atgcacatgt ttctgtgctt tcagattgca ataaatataa gacagagcac 360aagtttgatc ttgtgatatc tgatatgtat acagacaatg attcaaaaag aaagcatgaa 420ggcgtgatag ccaataatgg caatgatgac gttttcatat atctctcaag ttttcttcgt 480aataatttgg ctctaggtgg tagttttgct gtaaaagtga cagagacaag ttggcacgaa 540gttttatatg acattgcaca ggattgtgca tggtggacaa tgttttgtac agcagtgaat 600gcctcttctt cagaagcatt cttgattggt gttaattatt tgggtgcaag tgaaaaggtt 660aaggttagtg gaaaaacgct gcacgcaaat tatatatttt ggaggaattg taattattta 720caaacctctg cttatagtat atttgacgtt gctaagtttg atttgagatt gaaagcaacg 780ccagttgtta atttgaaaac tgaacaaaag acagacttag tctttaattt aattaagtgt 840ggtaagttac tggtaagaga tgttggtaac acctctttta ctagtgactc ttttgtgtgt 900actatgtag 9096145PRTInfectious bronchitis virus 6Ser Lys Gly His Glu Thr Glu Glu Val Asp Ala Val Gly Ile Leu Ser 1 5 10 15 Leu Cys Ser Phe Ala Val Asp Pro Ala Asp Thr Tyr Cys Lys Tyr Val 20 25 30 Ala Ala Gly Asn Gln Pro Leu Gly Asn Cys Val Lys Met Leu Thr Val 35 40 45 His Asn Gly Ser Gly Phe Ala Ile Thr Ser Lys Pro Ser Pro Thr Pro 50 55 60 Asp Gln Asp Ser Tyr Gly Gly Ala Ser Val Cys Leu Tyr Cys Arg Ala 65 70 75 80 His Ile Ala His Pro Gly Gly Ala Gly Asn Leu Asp Gly Arg Cys Gln 85 90 95 Phe Lys Gly Ser Phe Val Gln Ile Pro Thr Thr Glu Lys Asp Pro Val 100 105 110 Gly Phe Cys Leu Arg Asn Lys Val Cys Thr Val Cys Gln Cys Trp Ile 115 120 125 Gly Tyr Gly Cys Gln Cys Asp Ser Leu Arg Gln Pro Lys Pro Ser Val 130 135 140 Gln 145 7521PRTInfectious bronchitis virus 7Gly Thr Gly Leu Phe Lys Ile Cys Asn Lys Glu Phe Ser Gly Val His 1 5 10 15 Pro Ala Tyr Ala Val Thr Thr Lys Ala Leu Ala Ala Thr Tyr Lys Val 20 25 30 Asn Asp Glu Leu Ala Ala Leu Val Asn Val Glu Ala Gly Ser Glu Ile 35 40 45 Thr Tyr Lys His Leu Ile Ser Leu Leu Gly Phe Lys Met Ser Val Asn 50 55 60 Val Glu Gly Cys His Asn Met Phe Ile Thr Arg Asp Glu Ala Ile Arg 65 70 75 80 Asn Val Arg Gly Trp Val Gly Phe Asp Val Glu Ala Thr His Ala Cys 85 90 95 Gly Thr Asn Ile Gly Thr Asn Leu Pro Phe Gln Val Gly Phe Ser Thr 100 105 110 Gly Ala Asp Phe Val Val Thr Pro Glu Gly Leu Val Asp Thr Ser Ile 115 120 125 Gly Asn Asn Phe Glu Pro Val Asn Ser Lys Ala Pro Pro Gly Glu Gln 130 135 140 Phe Asn His Leu Arg Ala Leu Phe Lys Ser Ala Lys Pro Trp His Val 145 150 155 160 Val Arg Pro Arg Ile Val Gln Met Leu Ala Asp Asn Leu Cys Asn Val 165 170 175 Ser Asp Cys Val Val Phe Val Thr Trp Cys His Gly Leu Glu Leu Thr 180 185 190 Thr Leu Arg Tyr Phe Val Lys Ile Gly Lys Asp Gln Val Cys Ser Cys 195 200 205 Gly Ser Arg Ala Thr Thr Phe Asn Ser His Thr Gln Ala Tyr Ala Cys 210 215 220 Trp Lys His Cys Leu Gly Phe Asp Phe Val Tyr Asn Pro Leu Leu Val 225 230 235 240 Asp Ile Gln Gln Trp Gly Tyr Ser Gly Asn Leu Gln Phe Asn His Asp 245 250 255 Leu His Cys Asn Val His Gly His Ala His Val Ala Ser Ala Asp Ala 260 265 270 Ile Met Thr Arg Cys Leu Ala Ile Asn Asn Ala Phe Cys Gln Asp Val 275 280 285 Asn Trp Asp Leu Thr Tyr Pro His Ile Ala Asn Glu Asp Glu Val Asn 290 295 300 Ser Ser Cys Arg Tyr Leu Gln Arg Met Tyr Leu Asn Ala Cys Val Asp 305 310 315 320 Ala Leu Lys Val Asn Val Val Tyr Asp Ile Gly Asn Pro Lys Gly Ile 325 330 335 Lys Cys Val Arg Arg Gly Asp Leu Asn Phe Arg Phe Tyr Asp Lys Asn 340 345 350 Pro Ile Val Pro Asn Val Lys Gln Phe Glu Tyr Asp Tyr Asn Gln His 355 360 365 Lys Asp Lys Phe Ala Asp Gly Leu Cys Met Phe Trp Asn Cys Asn Val 370 375 380 Asp Cys Tyr Pro Asp Asn Ser Leu Val Cys Arg Tyr Asp Thr Arg Asn 385 390 395 400 Leu Ser Val Phe Asn Leu Pro Gly Cys Asn Gly Gly Ser Leu Tyr Val 405 410 415 Asn Lys His Ala Phe His Thr Pro Lys Phe Asp Arg Thr Ser Phe Arg 420 425 430 Asn Leu Lys Ala Met Pro Phe Phe Phe Tyr Asp Ser Ser Pro Cys Glu 435 440 445 Thr Ile Gln Leu Asp Gly Val Ala Gln Asp Leu Val Ser Leu Ala Thr 450 455 460 Lys Asp Cys Ile Thr Lys Cys Asn Ile Gly Gly Ala Val Cys Lys Lys 465 470 475 480 His Ala Gln Met Tyr Ala Asp Phe Val Thr Ser Tyr Asn Ala Ala Val 485 490 495 Thr Ala Gly Phe Thr Phe Trp Val Thr Asn Asn Phe Asn Pro Tyr Asn 500 505 510 Leu Trp Lys Ser Phe Ser Ala Leu Gln 515 520 8338PRTInfectious bronchitis virus 8Ser Ile Asp Asn Ile Ala Tyr Asn Met Tyr Lys Gly Gly His Tyr Asp 1 5 10 15 Ala Ile Ala Gly Glu Met Pro Thr Ile Val Thr Gly Asp Lys Val Phe 20 25 30 Val Ile Asp Gln Gly Val Glu Lys Ala Val Phe Phe Asn Gln Thr Ile 35 40 45 Leu Pro Thr Ser Val Ala Phe Glu Leu Tyr Ala Lys Arg Asn Ile Arg 50 55 60 Thr Leu Pro Asn Asn Arg Ile Leu Lys Gly Leu Gly Val Asp Val Thr 65 70 75 80 Asn Gly Phe Val Ile Trp Asp Tyr Thr Asn Gln Thr Pro Leu Tyr Arg 85 90 95 Asn Thr Val Lys Val Cys Ala Tyr Thr Asp Ile Glu Pro Asn Gly Leu 100 105 110 Ile Val Leu Tyr Asp Asp Arg Tyr Gly Asp Tyr Gln Ser Phe Leu Ala 115 120 125 Ala Asp Asn Ala Val Leu Val Ser Thr Gln Cys Tyr Lys Arg Tyr Ser 130 135 140 Tyr Val Glu Ile Pro Ser Asn Leu Leu Val Gln Asn Gly Ile Pro Leu 145 150 155 160 Lys Asp Gly Ala Asn Leu Tyr Val Tyr Lys Arg Val Asn Gly Ala Phe 165 170 175 Val Thr Leu Pro Asn Thr Leu Asn Thr Gln Gly Arg Ser Tyr Glu Thr 180 185 190 Phe Glu Pro Arg Ser Asp Val Glu Arg Asp Phe Leu Asp Met Ser Glu 195 200 205 Glu Ser Phe Val Glu Lys Tyr Gly Lys Glu Leu Gly Leu Gln His Ile 210 215 220 Leu Tyr Gly Glu Val Asp Lys Pro Gln Leu Gly Gly Leu His Thr Val 225 230 235 240 Ile Gly Met Cys Arg Leu Leu Arg Ala Asn Lys Leu Asn Ala Lys Ser 245 250 255 Val Thr Asn Ser Asp Ser Asp Val Met Gln Asn Tyr Phe Val Leu Ala 260 265 270 Asp Asn Gly Ser Tyr Lys Gln Val Cys Thr Val Val Asp Leu Leu Leu 275 280 285 Asp Asp Phe Leu Glu Leu Leu Arg Asn Ile Leu Lys Glu Tyr Gly Thr 290 295 300 Asn Lys Ser Lys Val Val Thr Val Ser Ile Asp Tyr His Ser Ile Asn 305 310 315 320 Phe Met Thr Trp Phe Glu Asp Gly Ile Ile Lys Thr Cys Tyr Pro Gln 325 330 335 Leu Gln 9302PRTInfectious bronchitis virus 9Ser Ala Trp Thr Cys Gly Tyr Asn Met Pro Glu Leu Tyr Lys Val Gln 1 5 10 15 Asn Cys Val Met Glu Pro Cys Asn Ile Pro Asn Tyr Gly Val Gly Ile 20 25 30 Ala Leu Pro Ser Gly Ile Met Met Asn Val Ala Lys Tyr Thr Gln Leu 35 40 45 Cys Gln Tyr Leu Ser Lys Thr Thr Met Cys Val Pro His Asn Met Arg 50 55 60 Val Met His Phe Gly Ala Gly Ser Asp Lys Gly Val Ala Pro Gly Ser 65 70 75 80 Thr Val Leu Lys Gln Trp Leu Pro Glu Gly Thr Leu Leu Val Asp Asn 85 90 95 Asp Ile Val Asp Tyr Val Ser Asp Ala His Val Ser Val Leu Ser Asp 100 105 110 Cys Asn Lys Tyr Lys Thr Glu His Lys Phe Asp Leu Val Ile Ser Asp 115 120 125 Met Tyr Thr Asp Asn Asp Ser Lys Arg Lys His Glu Gly Val Ile Ala 130 135 140 Asn Asn Gly Asn Asp Asp Val Phe Ile Tyr Leu Ser Ser Phe Leu Arg 145 150 155 160 Asn Asn Leu Ala Leu Gly Gly Ser Phe Ala Val Lys Val Thr Glu Thr 165 170 175 Ser Trp His Glu Val Leu Tyr Asp Ile Ala Gln Asp Cys Ala Trp Trp 180 185 190 Thr Met Phe Cys Thr Ala Val Asn Ala Ser Ser Ser Glu Ala Phe Leu 195 200 205 Val Gly Val Asn Tyr Leu Gly Ala Ser Glu Lys Val Lys Val Ser Gly 210 215 220 Lys Thr Leu His Ala Asn Tyr Ile Phe Trp Arg Asn Cys Asn Tyr Leu 225 230 235 240 Gln Thr Ser Ala Tyr Ser Ile Phe Asp Val Ala Lys Phe Asp Leu Arg 245 250 255 Leu Lys Ala Thr Pro Val Val Asn Leu Lys Thr Glu Gln Lys Thr Asp 260 265 270 Leu Val Phe Asn Leu Ile Lys Cys Gly Lys Leu Leu Val Arg Asp Val 275 280 285 Gly Asn Thr Ser Phe Thr Ser Asp Ser Phe Val Cys Thr Met 290 295 300 10145PRTArtificial SequenceMutated Nsp10 sequence 10Ser Lys Gly His Glu Thr Glu Glu Val Asp Ala Val Gly Ile Leu Ser 1 5 10 15 Leu Cys Ser Phe Ala Val Asp Pro Ala Asp Thr Tyr Cys Lys Tyr Val 20 25 30 Ala Ala Gly Asn Gln Pro Leu Gly Asn Cys Val Lys Met Leu Thr Val 35 40 45 His Asn Gly Ser Gly Phe Ala Ile Thr Ser Lys Pro Ser Pro Thr Pro 50 55 60 Asp Gln Asp Ser Tyr Gly Gly Ala Ser Val Cys Leu Tyr Cys Arg Ala 65 70 75 80 His Ile Ala His Leu Gly Gly Ala Gly Asn Leu Asp Gly Arg Cys Gln 85 90 95 Phe Lys Gly Ser Phe Val Gln Ile Pro Thr Thr Glu Lys Asp Pro Val 100 105 110 Gly Phe Cys Leu Arg Asn Lys Val Cys Thr Val Cys Gln Cys Trp Ile 115 120 125 Gly Tyr Gly Cys Gln Cys Asp Ser Leu Arg Gln Pro Lys Pro Ser Val 130 135 140 Gln 145 11521PRTArtificial SequenceMutated Nsp14 sequence 11Gly Thr Gly Leu Phe Lys Ile Cys Asn Lys Glu Phe Ser Gly Val His 1 5 10 15 Pro Ala Tyr Ala Val Thr Thr Lys Ala Leu Ala Ala Thr Tyr Lys Val 20 25 30 Asn Asp Glu Leu Ala Ala Leu Val Asn Val Glu Ala Gly Ser Glu Ile 35 40 45 Thr Tyr Lys His Leu Ile Ser Leu Leu Gly Phe Lys Met Ser Val Asn 50 55 60 Val Glu Gly Cys His Asn Met Phe Ile Thr Arg Asp Glu Ala Ile Arg 65 70 75 80 Asn Val Arg Gly Trp Val Gly Phe Asp Val Glu Ala Thr His Ala Cys 85 90 95 Gly Thr Asn Ile Gly Thr Asn Leu Pro Phe Gln Val Gly Phe Ser Thr 100 105 110 Gly Ala Asp Phe Val Val Thr Pro Glu Gly Leu Val Asp Thr Ser Ile 115 120 125 Gly Asn Asn Phe Glu Pro Val Asn Ser Lys Ala Pro Pro Gly Glu Gln 130 135 140 Phe Asn His Leu Arg Ala Leu Phe Lys Ser Ala Lys Pro Trp His Val 145 150 155 160 Val Arg Pro Arg Ile Val Gln Met Leu Ala Asp Asn Leu Cys Asn Val 165 170 175 Ser Asp Cys Val Val Phe Val Thr Trp Cys His Gly Leu Glu Leu Thr 180 185 190 Thr Leu Arg Tyr Phe Val Lys Ile Gly Lys Asp Gln Val Cys Ser Cys 195 200 205 Gly Ser Arg Ala Thr Thr Phe Asn Ser His Thr Gln Ala Tyr Ala Cys 210 215 220 Trp Lys His Cys Leu Gly Phe Asp Phe Val Tyr Asn Pro Leu Leu Val 225 230 235 240 Asp Ile Gln Gln Trp Gly Tyr Ser Gly Asn Leu Gln Phe Asn His Asp 245 250 255 Leu His Cys Asn Val His Gly His Ala His Val Ala Ser Ala Asp Ala 260 265 270 Ile Met Thr Arg Cys Leu Ala Ile Asn Asn Ala Phe Cys Gln Asp Val 275 280 285 Asn Trp Asp Leu Thr Tyr Pro His Ile Ala Asn Glu Asp Glu Val Asn 290 295 300 Ser Ser Cys Arg Tyr Leu Gln Arg Met Tyr Leu Asn Ala Cys Val Asp 305 310 315 320 Ala Leu Lys Val Asn Val Val Tyr Asp Ile Gly Asn Pro Lys Gly Ile 325 330 335 Lys Cys Val Arg Arg Gly Asp Leu Asn Phe Arg Phe Tyr Asp Lys Asn 340 345 350 Pro Ile Val Pro Asn Val Lys Gln Phe Glu Tyr Asp Tyr Asn Gln His 355 360 365 Lys Asp Lys Phe Ala Asp Gly Leu Cys Met Phe Trp Asn Cys Asn Val 370 375 380 Asp Cys Tyr Pro Asp Asn Ser Leu Leu Cys Arg Tyr Asp Thr Arg Asn 385 390 395 400 Leu Ser Val Phe Asn Leu Pro Gly Cys Asn Gly Gly Ser Leu Tyr Val 405 410 415 Asn Lys His Ala Phe His Thr Pro Lys Phe Asp Arg Thr Ser Phe Arg 420 425 430 Asn Leu Lys Ala Met Pro Phe Phe Phe Tyr Asp Ser Ser Pro Cys Glu 435 440 445 Thr Ile Gln Leu Asp Gly Val Ala Gln Asp Leu Val Ser Leu Ala Thr 450 455 460 Lys Asp Cys Ile Thr Lys Cys Asn Ile Gly Gly Ala Val Cys Lys Lys 465 470 475 480 His Ala Gln Met Tyr Ala Asp Phe Val Thr Ser Tyr Asn Ala Ala Val 485 490 495 Thr Ala Gly Phe Thr Phe Trp Val Thr Asn Asn Phe Asn Pro Tyr Asn 500 505 510 Leu Trp Lys Ser Phe Ser Ala Leu Gln 515 520 12338PRTArtificial SequenceMutated Nsp15 sequence 12Ser Ile Asp Asn Ile Ala Tyr Asn Met Tyr Lys Gly Gly His Tyr Asp 1 5 10 15 Ala Ile Ala Gly Glu Met Pro Thr Ile Val Thr Gly Asp Lys Val Phe 20 25 30 Val Ile Asp Gln Gly Val Glu Lys Ala Val Phe Phe Asn Gln Thr
Ile 35 40 45 Leu Pro Thr Ser Val Ala Phe Glu Leu Tyr Ala Lys Arg Asn Ile Arg 50 55 60 Thr Leu Pro Asn Asn Arg Ile Leu Lys Gly Leu Gly Val Asp Val Thr 65 70 75 80 Asn Gly Phe Val Ile Trp Asp Tyr Thr Asn Gln Thr Pro Leu Tyr Arg 85 90 95 Asn Thr Val Lys Val Cys Ala Tyr Thr Asp Ile Glu Pro Asn Gly Leu 100 105 110 Ile Val Leu Tyr Asp Asp Arg Tyr Gly Asp Tyr Gln Ser Phe Leu Ala 115 120 125 Ala Asp Asn Ala Val Leu Val Ser Thr Gln Cys Tyr Lys Arg Tyr Ser 130 135 140 Tyr Val Glu Ile Pro Ser Asn Leu Leu Val Gln Asn Gly Ile Pro Leu 145 150 155 160 Lys Asp Gly Ala Asn Leu Tyr Val Tyr Lys Arg Val Asn Gly Ala Phe 165 170 175 Val Thr Leu Pro Asn Thr Ile Asn Thr Gln Gly Arg Ser Tyr Glu Thr 180 185 190 Phe Glu Pro Arg Ser Asp Val Glu Arg Asp Phe Leu Asp Met Ser Glu 195 200 205 Glu Ser Phe Val Glu Lys Tyr Gly Lys Glu Leu Gly Leu Gln His Ile 210 215 220 Leu Tyr Gly Glu Val Asp Lys Pro Gln Leu Gly Gly Leu His Thr Val 225 230 235 240 Ile Gly Met Cys Arg Leu Leu Arg Ala Asn Lys Leu Asn Ala Lys Ser 245 250 255 Val Thr Asn Ser Asp Ser Asp Val Met Gln Asn Tyr Phe Val Leu Ala 260 265 270 Asp Asn Gly Ser Tyr Lys Gln Val Cys Thr Val Val Asp Leu Leu Leu 275 280 285 Asp Asp Phe Leu Glu Leu Leu Arg Asn Ile Leu Lys Glu Tyr Gly Thr 290 295 300 Asn Lys Ser Lys Val Val Thr Val Ser Ile Asp Tyr His Ser Ile Asn 305 310 315 320 Phe Met Thr Trp Phe Glu Asp Gly Ile Ile Lys Thr Cys Tyr Pro Gln 325 330 335 Leu Gln 13302PRTArtificial SequenceMutated Nsp16 sequence 13Ser Ala Trp Thr Cys Gly Tyr Asn Met Pro Glu Leu Tyr Lys Val Gln 1 5 10 15 Asn Cys Val Met Glu Pro Cys Asn Ile Pro Asn Tyr Gly Val Gly Ile 20 25 30 Ala Leu Pro Ser Gly Ile Met Met Asn Val Ala Lys Tyr Thr Gln Leu 35 40 45 Cys Gln Tyr Leu Ser Lys Thr Thr Met Cys Val Pro His Asn Met Arg 50 55 60 Val Met His Phe Gly Ala Gly Ser Asp Lys Gly Val Ala Pro Gly Ser 65 70 75 80 Thr Val Leu Lys Gln Trp Leu Pro Glu Gly Thr Leu Leu Val Asp Asn 85 90 95 Asp Ile Val Asp Tyr Val Ser Asp Ala His Val Ser Val Leu Ser Asp 100 105 110 Cys Asn Lys Tyr Lys Thr Glu His Lys Phe Asp Leu Val Ile Ser Asp 115 120 125 Met Tyr Thr Asp Asn Asp Ser Lys Arg Lys His Glu Gly Val Ile Ala 130 135 140 Asn Asn Gly Asn Asp Asp Val Phe Ile Tyr Leu Ser Ser Phe Leu Arg 145 150 155 160 Asn Asn Leu Ala Leu Gly Gly Ser Phe Ala Val Lys Val Thr Glu Thr 165 170 175 Ser Trp His Glu Val Leu Tyr Asp Ile Ala Gln Asp Cys Ala Trp Trp 180 185 190 Thr Met Phe Cys Thr Ala Val Asn Ala Ser Ser Ser Glu Ala Phe Leu 195 200 205 Ile Gly Val Asn Tyr Leu Gly Ala Ser Glu Lys Val Lys Val Ser Gly 210 215 220 Lys Thr Leu His Ala Asn Tyr Ile Phe Trp Arg Asn Cys Asn Tyr Leu 225 230 235 240 Gln Thr Ser Ala Tyr Ser Ile Phe Asp Val Ala Lys Phe Asp Leu Arg 245 250 255 Leu Lys Ala Thr Pro Val Val Asn Leu Lys Thr Glu Gln Lys Thr Asp 260 265 270 Leu Val Phe Asn Leu Ile Lys Cys Gly Lys Leu Leu Val Arg Asp Val 275 280 285 Gly Asn Thr Ser Phe Thr Ser Asp Ser Phe Val Cys Thr Met 290 295 300
* * * * *
from sun up to sun down, and you two turned down any money as payment in exchange for pocus ass weed, white hugs and pats on the heads. All Killabref wanted a pat on his ass from his massa... 
