Modified S2 subunit of coronavirus spike protein

Abstract

The present invention relates to a recombinant avian coronavirus spike protein or a fragment thereof, comprising a mutation at amino acid position 865. Further, the present invention relates to an immunogenic composition comprising an avian coronavirus having such a spike protein.

Description

[0001] sequence list

[0002] This application contains sequence listings in accordance with 37C.FR1.821–1.825. The sequence listings accompanying this application are incorporated herein by reference in their entirety. Background Technology

[0003] Avian infectious bronchitis virus (IBV) is a protozoan belonging to the order Nidovirales and family Coronaviridae. IBV primarily infects the upper respiratory tract epithelium of chickens, causing respiratory disease, often secondary to bacterial pathogens (Cook et al. 2012. Avian Pathol. 41:239-250). Some IBV strains also affect the renal tubules, oviducts, and parts of the gastrointestinal tract, leading to pathological damage and clinical symptoms in these organ systems. The virus is present worldwide in both commercial and free-range chickens. Due to its high genomic variability, IBV is classified into a wide variety of genotypes, serotypes, and protective types. IBV is currently considered one of the most economically relevant viral pathogens in the poultry industry.

[0004] Infectious bronchitis virus (IBV) is an enveloped virus with a 27.6 kb positive-sense single-stranded RNA genome (Cavanagh 2007. Vet. Res. 38:281-297). The first two-thirds of the viral genome contains a large coding region (also designated as gene 1), divided into two open reading frames 1a and 1b, which encodes at least 15 non-structural proteins involved in RNA cell tropism, editing, and transcription. The latter third of the viral genome encodes structural proteins: the spike protein (S, encoded by gene 2), the envelope protein (E, encoded by gene 3c), the membrane protein (M, encoded by gene 4), and the nucleocapsid protein (N, encoded by gene 6). Proteins S, E, and M form the viral envelope, while protein N, along with the viral RNA, forms the ribonucleoprotein core. The coronavirus spike protein determines host species tropism (Kuo et al. 2000. J. Virol. 74:1393-1406). It is a dimeric or trimeric transmembrane protein that is cleaved by proteases into two subunits, S1 and S2. The glycosylated S1 domain forms the 'head' of the spike protein and contains a receptor-binding domain that interacts with 2,3-linked sialic acid on the surface of the host cell (Promkuntod et al. 2014. Virology. 448:26-32). The S2 domain contains the remainder of the extracellular domain ('stem'), a transmembrane domain, and an intracellular domain located in the cytoplasm.

[0005] The widely used attenuated live IBV vaccine strains H52 and H120 were developed in the Netherlands in the 1960s through continuous passage of the Massachusetts serotype IBV strain in embryo-containing eggs (Bijlenga et al. 2004; Avian Pathol. 33:550-557). These vaccine strains must also be propagated in embryo-containing eggs for production. Currently, IBV vaccines (both inactivated and live vaccines) are still propagated in embryo-containing eggs, which is cumbersome and expensive.

[0006] The only IBV adapted to cell lines described to date is the IBV strain Beaudette, which replicates efficiently in Vero and BHK cells. Casais et al. (2003) generated recombinant IBV using the extracellular domain sequence of the Beaudette spike, demonstrating that the Beaudette S protein is the determinant of cell line tropism, capable of transferring extended cell line tropism to another IBV (M41). WO 2011 / 004146 discloses that the S2 subunit from Beaudette is responsible for extended tissue tropism. The sequence within the S2 subunit, the heparan sulfate binding site from Beaudette, has been identified as responsible for extended cell line tropism. Furthermore, Bickerton et al. (2018) (Journal of Virology 92(19)) disclosed an eight-amino acid Beaudette-specific motif. However, recombinant IBV with the Beaudette spike S2 ​​subunit is unsuitable as a vaccine. Ellis et al. 2018 (J.Virol.92(23)) described that recombinant Beaudette with chimeric spikes does not provide adequate protection against homologous S1 attack, which has heterologous S1 subunits from M41 or QX combined with the S2 subunits of the Beaudette spike. In addition, wild-type Beaudette does not provide protection against homologous attack like other licensed vaccines belonging to the Massachusetts serotype (Hodgson et al. 2004: J Virol 78:13804–13811 or Geilhausen et al. 1973: Archiv für die gesamte Virusforschung 40:285-290).

[0007] Fang et al. (2005, Biochemical and Biophysical Research Communication 336; pp. 417-423) disclosed that Beaudette's adaptation to proliferation in Vero cells resulted in 49 amino acid modifications, 26 of which were localized within the spike protein.

[0008] In summary, providing an IBV vaccine with extended cell culture or tissue tropism by exchanging the spike protein for a heterologous Beaudette spike protein does not result in an IBV vaccine providing sufficient efficacy, and using the Beaudette spike sequence will limit protection against Massachusetts serotypes and lack cross-protection against further genotypes. Furthermore, prior art motifs or sites identified in Beaudette have not been transferred into IBV vaccines that exhibit both extended cell culture or tissue tropism and protective efficacy (interference between extended tropism and vaccine efficacy has not yet been shown).

[0009] Therefore, a single amino acid or short motif is needed that can be transferred into IBV or IBV vaccines without affecting vaccine efficacy, but rather to enable extended cell tropism or tissue tropism. Detailed Implementation

[0010] Before describing aspects of the invention, it must be noted that, as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include the plural referents unless the context clearly indicates otherwise. Thus, for example, reference to “an antigen” includes multiple antigens, reference to “virus” refers to one or more viruses and their equivalents known to those skilled in the art, and so on. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although preferred methods, apparatus, and materials are now described, any methods and materials similar to or equivalent to those described herein may be used in the practice or testing of the invention. All publications mentioned herein are incorporated by reference for the purpose of describing and disclosing cell lines, vectors, and methods as reported in the publications that can be used in conjunction with the invention. Nothing herein should be construed as an admission that the invention is not entitled to precede such disclosure by prior invention.

[0011] Composition of matter

[0012] This invention solves the inherent problems in the prior art and provides a significant advancement in the current level of technology.

[0013] Generally, the present invention provides an avian coronavirus spike protein or a fragment thereof containing an aromatic amino acid at position 865, wherein the spike protein or the fragment thereof is not derived from IBV strain M41.

[0014] Furthermore, the present invention provides an avian coronavirus spike protein or a fragment thereof, wherein at least a portion of the S2 subunit is derived from an avian coronavirus having limited cell tropism or tissue tropism, and wherein the amino acid at position 865 is an aromatic amino acid.

[0015] Furthermore, the present invention provides a recombinant avian coronavirus spike protein or a fragment thereof containing a mutation at amino acid position 865.

[0016] Generally, the present invention provides an IBV spike protein or a fragment thereof containing an aromatic amino acid at position 865, wherein the spike protein or the fragment thereof is not derived from the IBV strain M41.

[0017] The present invention also provides an IBV spike protein or a fragment thereof, wherein at least a portion of the S2 subunit is derived from IBV having limited cell tropism or tissue tropism, and wherein the amino acid at position 865 is an aromatic amino acid.

[0018] Furthermore, the present invention provides a recombinant IBV spike protein or a fragment thereof containing a mutation at amino acid position 865.

[0019] Advantageously, experimental data showed that modifying a single site, position 865, within the spike protein with an aromatic amino acid (histidine) gave coronavirus strains (such as exemplary H52 and CR88 IBV strains) expanded cell tropism or tissue tropism.

[0020] The term "coronavirus" is well known to those skilled in the art. Generally, coronaviruses are viruses belonging to the subfamily Coronavirinae of the family Coronaviridae in the order Nidovirales. Coronaviruses are enveloped viruses and possess a positive-sense, single-stranded RNA genome containing a helical nucleocapsid. The term "coronavirus" includes all strains, genotypes, protective types, and serotypes of infectious bronchitis virus. Examples of avian coronaviruses are infectious bronchitis virus (IBV); guinea fowl coronavirus (GfCoV) and turkey coronavirus (TCoV; turkey enteritis virus and blue crown virus).

[0021] The term "IBV" refers to infectious bronchitis virus, as is well known to those skilled in the art. The term "IBV" includes all strains, genotypes, protective types, and serotypes of infectious bronchitis virus.

[0022] The term "mutation" encompasses modifications in proteins encoded by viral RNA that result in alterations to said encoded protein. Further, the term "mutation" includes genetically modified mutations. The term mutation involves, but is not limited to, substitution (the replacement of one or more nucleotide / base pairs), deletion (the removal of one, several, or all nucleotide / base pairs), and / or insertion (the addition of one or more nucleotide / base pairs). As used herein, a mutation can be a single mutation or several mutations; therefore, the term "one or more mutations" is frequently used and encompasses both single and several mutations. However, the term mutation is well-known to those skilled in the art, and mutations can be generated effortlessly by those skilled in the art.

[0023] The term "spike" refers to a specific protein of avian coronavirus or IBV, well known to those skilled in the art. The spike protein is a major inducer of antibody and protective immune responses. Furthermore, the spike (S) protein promotes cellular entry of avian coronavirus or IBV by binding to cell receptors on host cells and by mediating virus-cell membrane fusion with the host cell membrane. Additionally, it determines the cell tropism and tissue tropism of the viral strain.

[0024] The terms "protein," "amino acid," and "peptide" are used interchangeably. The term "protein" refers to an amino acid sequence consisting of naturally occurring amino acids and their derivatives. Naturally occurring amino acids are well-known in the art and are described in standard biochemistry textbooks. Within an amino acid sequence, amino acids are linked by peptide bonds. Further, the two ends of the amino acid sequence are referred to as the carboxyl terminus (C-terminus) and amino terminus (N-terminus). The term "protein" includes substantially purified proteins or protein preparations that additionally contain other proteins. Further, the term also refers to protein fragments. Additionally, it includes chemically modified proteins. Such modifications can be artificial or naturally occurring, such as phosphorylation, glycosylation, tetradecylation, etc.

[0025] Mutation 865

[0026] In one aspect of the avian coronavirus or IBV spike protein or a fragment thereof according to the invention, an aromatic amino acid at amino acid position 865 is introduced by mutation. The term "introduced" means that the mutation has been introduced by genetic engineering (artificially, e.g., through human intervention).

[0027] In another specific aspect of the avian coronavirus or IBV spike protein or fragment thereof according to the invention, mutations are amino acid substitutions, deletions, or insertions.

[0028] In another specific aspect of the avian coronavirus or IBV spike protein or fragment thereof according to the invention, an amino acid at amino acid position 865 is mutated to an aromatic amino acid; or a polar amino acid at amino acid position 865 is mutated to an aromatic amino acid; or glutamine at amino acid position 865 is mutated to histidine.

[0029] In another specific aspect of the avian coronavirus or IBV spike protein or fragment thereof according to the invention, the aromatic amino acid at amino acid position 865 is histidine.

[0030] Extended cell tropism or tissue tropism

[0031] In another specific aspect of the avian coronavirus or IBV spike protein or fragment thereof according to the invention, an aromatic amino acid at amino acid position 865 or the mutation at amino acid position 865 results in extended cell tropism or tissue tropism of the avian coronavirus or IBV.

[0032] The term "cell or tissue" is known to those skilled in the art. The term "cell" includes cell lines, such as those listed elsewhere herein, and primary cells. The term "tissue" includes cells derived from tissues, such as those listed elsewhere herein, exemplarily, primary chicken embryo cells or primary chicken fibroblasts from the lung or liver. This term includes the propagation of cells or tissues (cells) in culture outside of an organism. The term "culture" refers to the propagation of cells (e.g., cell lines, primary cells, or tissue cells) outside of an organism under defined culture conditions known to those skilled in the art.

[0033] The term "extended tropism" means that the avian coronavirus or IBV of the present invention can reproduce in cells (e.g., cell lines) or tissue cells (other than primary chicken embryo cells from the kidney). In contrast, coronavirus vaccines (e.g., IBV vaccines) or non-cell culture-adapted wild-type coronaviruses or IBV (describing the cell line-adapted IBV Beaudette strain) can only reproduce in embryo-containing eggs or primary chicken embryo cells from the kidney (after adaptation). Accordingly, the coronaviruses of the present invention (e.g., IBV) having extended cell tropism or tissue tropism have the ability to infect and / or replicate in one or more cell lines or tissue cells other than primary chicken embryo cells from the kidney. Preferably, the coronaviruses of the present invention (e.g., IBV) having extended cell tropism or tissue tropism have the ability to infect and / or replicate in one or more cell lines as listed herein. Accordingly, for example, coronaviruses or IBV having extended cell tropism or tissue tropism may have the ability to infect and / or replicate in BHK or EB66 cells.

[0034] The term "limited tropism" means that avian coronaviruses or IBV can only reproduce in primary chicken embryo cells derived from the kidneys under any circumstances. Accordingly, coronaviruses or IBVs with limited cell tropism or tissue tropism do not have the ability to infect and / or replicate in cells such as BHK or EB66.

[0035] Advantageously, experimental data showed that after modifying a single amino acid position 865 within the spike protein to an aromatic amino acid, IBV strains, exemplarily H52 and CR88, exhibit expanded cell or tissue tropism. Furthermore, it has been shown that the modification to histidine at position 865 is genetically stable.

[0036] In another specific aspect of the avian coronavirus or IBV spike protein or fragments thereof according to the invention, avian coronavirus or IBV infects and / or replicates in at least one cell line or cell selected from the following list: primary chicken embryo cells or primary chicken fibroblasts from the lung or liver, chicken embryo fibroblast cell lines, duck embryo stem cell lines, human embryo kidney cell lines, young hamster kidney cell lines, African green monkey kidney cell lines, rabbit kidney cell lines, canine kidney cell lines, chicken liver cell lines, bovine kidney cell lines, porcine kidney cell lines, and insect cell lines.

[0037] In another specific aspect of the avian coronavirus or IBV spike protein or fragments thereof according to the invention, avian coronavirus or IBV is infected and / or replicated in at least one cell line selected from the following list: DF-1 (Douglas Foster), EB66 (duck embryo stem cell line), PBS-12, PBS-12SF (serum-free PBS-12), BHK21 (juvenile hamster kidney), HEK 293T (human embryo kidney), Vero (Verda Reno), MA104, RK13 (rabbit kidney), LMH (leghorn male liver cancer), MDCK (Madin-Darby canine kidney), MDBK (Madin-Darby bovine kidney), PK15 (porcine kidney), PK2A (porcine kidney), SF9, SF21, and SF+ (fall armyworm (Spodoptera frugiperda)).

[0038] In another specific aspect of the avian coronavirus or IBV spike protein or fragment thereof according to the invention, avian coronavirus or IBV is infected and / or replicated in at least one cell line selected from the following list: DF-1, EB66, PBS-12, PBS-12SF, BHK, HEK 293T, Vero, MA104 and RK13.

[0039] Preferably, IBV infects and / or replicates in EB66 or BHK cell lines.

[0040] All cell lines mentioned are well known to those skilled in the art and are commercially available and / or publicly available. MDCK cells are exemplarily deposited at the American Tissue Culture Collection (ATCC) under accession number ATCC CCL-34 or ATCC CRL-2285. DF-1 cells are exemplarily deposited at the American Tissue Culture Collection (ATCC) under accession number ATCC CRL-12203. PBS-12SF cells are exemplarily deposited at the American Tissue Culture Collection (ATCC) under accession number ATCC PTA-8565, or at RRID under CVCL_1K17. BHK-21 cells are exemplarily deposited at the American Tissue Culture Collection (ATCC) under accession number ATCC CCL-10. HEK 293T cells are exemplarily deposited at the American Tissue Culture Collection (ATCC) under accession number ATCC CRL-3216. Vero cells are exemplarily deposited at the American Tissue Culture Collection (ATCC) under accession number ATCC CCL-81. MA104 cells are exemplary deposited at the U.S. Center for Tissue Cultures under accession number ATCC CRL-2378. RK13 cells are exemplary deposited at the U.S. Center for Tissue Cultures under accession number ATCCCCL-37.

[0041] Amino acid position 865 numbering

[0042] In another specific aspect of the avian coronavirus or IBV spike protein or fragment thereof according to the invention, the designation of amino acid position 865 refers to amino acid position 865 in the spike protein of IBV H52, IBV H120 or M41.

[0043] In another specific aspect of the avian coronavirus or IBV spike protein or fragment thereof according to the invention, the designation of amino acid position 865 refers to amino acid position 865 in the spike protein of IBV H52.

[0044] In another specific aspect of the avian coronavirus or IBV spike protein or fragment thereof according to the invention, the designation of amino acid position 865 refers to amino acid position 865 in the spike protein as exemplarily given in SEQ ID NO:1.

[0045] In another specific aspect of the avian coronavirus or IBV spike protein or fragments thereof according to the invention, the amino acid sequence of SEQ ID NO:1 is used to determine the position number in the spike protein.

[0046] In another specific aspect of the avian coronavirus or IBV spike protein or fragment thereof according to the invention, in order to determine the amino position 865 in the spike protein, the amino acid sequence is compared with the amino acid sequence of SEQ ID NO:1.

[0047] In another specific aspect of the avian coronavirus or IBV spike protein or fragment thereof according to the invention, amino acid position 865 is located within the S2 subunit of the spike protein.

[0048] In another specific aspect of the avian coronavirus or IBV spike protein or fragment thereof according to the invention, amino acid position 865 corresponds to amino acid position 867 of the spike protein sequence of IBV CR88.

[0049] In another specific aspect of the avian coronavirus or IBV spike protein or fragment thereof according to the invention, amino acid position 865 corresponds to amino acid position 868 of the spike protein sequence of IBV QX.

[0050] In another specific aspect of the avian coronavirus or IBV spike protein or fragment thereof according to the invention, amino acid position 865 corresponds to amino acid position 869 of the spike protein sequence of IBV Q1.

[0051] In another specific aspect of the avian coronavirus or IBV spike protein or fragment thereof according to the invention, amino acid position 865 corresponds to amino acid position 868 of the spike protein sequence of IBV Var2.

[0052] In another specific aspect of the avian coronavirus or IBV spike protein or fragment thereof according to the invention, amino acid position 865 corresponds to amino acid position 872 of the spike protein sequence of IBV Brazil.

[0053] In another specific aspect of the avian coronavirus or IBV spike protein or fragment thereof according to the invention, amino acid position 865 corresponds to amino acid position 871 of the spike protein sequence of IBV Ark99.

[0054] In another specific aspect of the avian coronavirus or IBV spike protein or fragment thereof according to the invention, the spike protein has one or more amino acids selected from the following:

[0055] -862 is alanine, and / or

[0056] -864 is alanine, and / or

[0057] -866 is valine, and / or

[0058] -867 is aspartic acid, and / or

[0059] -868 is arginine.

[0060] The amino acid position number refers to the amino acid position within the spike protein, as exemplarily given in SEQ ID NO:1.

[0061] spike

[0062] The present invention also provides the spike protein or fragment thereof as described above, wherein the spike protein or fragment thereof is selected from: infectious bronchitis virus (IBV); guinea fowl coronavirus (GfCoV), turkey coronavirus (TCoV; turkey enteritis virus and blue crown virus), feline infectious peritonitis virus (FIPV), feline enteric coronavirus (FECV); infectious gastroenteritis virus (TGEV), porcine respiratory coronavirus (PRCoV), porcine epidemic diarrhea virus (PEDV), porcine hemagglutinating encephalomyelitis virus (PHEV); severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), human coronavirus 229E (HCoV-229E), human coronavirus NL63 (HCoV-NL63), human coronavirus HKU1 (HCoV-HKU1), human coronavirus OC43 (HCoV-OC43); canine coronavirus (CCoV), canine respiratory coronavirus (CRCoV), mouse hepatitis virus (MHV), and bovine coronavirus (BCV). Therefore, the present invention also provides a coronavirus spike protein or a fragment thereof, wherein at least a portion of the S2 subunit is derived from a coronavirus having limited cell tropism or tissue tropism, and wherein an aromatic amino acid is present at amino acid position 865, and wherein the spike protein is selected from: infectious bronchitis virus (IBV); guinea fowl coronavirus (GfCoV), turkey coronavirus (TCoV; turkey enteritis virus and blue crown virus), feline infectious peritonitis virus (FIPV), feline enteric coronavirus (FECV); infectious gastroenteritis virus (TGEV), porcine respiratory coronavirus (PRCoV), and porcine influenza virus. Evanescent diarrhea virus (PEDV), porcine hemagglutinating encephalomyelitis virus (PHEV); severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), human coronavirus 229E (HCoV-229E), human coronavirus NL63 (HCoV-NL63), human coronavirus HKU1 (HCoV-HKU1), human coronavirus OC43 (HCoV-OC43); canine coronavirus (CCoV), canine respiratory coronavirus (CRCoV), mouse hepatitis virus (MHV), bovine coronavirus (BCV).Furthermore, the present invention also provides a recombinant coronavirus spike protein or a fragment thereof, comprising a mutation at amino acid position 865, wherein the spike protein is selected from: infectious bronchitis virus (IBV); guinea fowl coronavirus (GfCoV), turkey coronavirus (TCoV; turkey enteritis virus and blue crown virus), feline infectious peritonitis virus (FIPV), feline enteric coronavirus (FECV); infectious gastroenteritis virus (TGEV), porcine respiratory coronavirus (PRCoV), porcine epidemic diarrhea virus (PEDV), porcine hemagglutinating encephalomyelitis virus (PHEV); severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), human coronavirus 229E (HCoV-229E), human coronavirus NL63 (HCoV-NL63), human coronavirus HKU1 (HCoV-HKU1), human coronavirus OC43 (HCoV-OC43); canine coronavirus (CCoV), canine respiratory coronavirus (CRCoV), mouse hepatitis virus (MHV), and bovine coronavirus (BCV).

[0063] In another specific aspect of the avian coronavirus spike protein or a fragment thereof according to the invention, the avian coronavirus spike protein or a fragment thereof is selected from: infectious bronchitis virus (IBV); guinea fowl coronavirus (GfCoV); and turkey coronavirus (TCoV; turkey enteritis virus and blue crown virus).

[0064] In another specific aspect of the avian coronavirus spike protein or fragments thereof according to the invention, the avian coronavirus is IBV (infectious bronchitis virus).

[0065] Infectious bronchitis virus (IBV) strains can be classified by serotype and genotype. Serotype classification involves treating the virus with neutralizing antibodies, while genotype classification generally involves examining the sequence of the S1 (spike) protein. However, different IBV strains are well known to those skilled in the art. Infectious bronchitis virus was first discovered in the United States in the 1930s.

[0066] The first IBV serotype identified was Massachusetts, and it remained the only serotype until the discovery of other IBV serotypes in 1956. Today, in addition to the originally identified Massachusetts type, several other serotypes have been identified in the United States, including Arkansas and Delaware. IBV Mass virus can now be identified in many countries worldwide.

[0067] The IBV strain Beaudette is a Massachusetts serotype and was derived after at least 150 passages in chicken embryos. The Beaudette IBV strain was originally isolated by Beaudette and Hudson (J. Am. Vet. Med. A. 90, 51-60, 1937) and passaged in chicken embryos. Other Massachusetts serotypes of IBV besides Beaudette are H120, H52, and M41. The H120 strain was passaged 120 times in embryo-containing eggs.

[0068] IBV QX has been described as a highly virulent wild isolate of IBV originally isolated in China. However, the virus has spread to Europe and has been identified in parts of Western Europe, primarily in the Netherlands, as well as in Germany, France, Belgium, Denmark, and the United Kingdom. Additionally, the QX genotype or serotype has been described in several countries in Asia and Africa.

[0069] The strain designated "Italien-02" or "Italy-02" was isolated in Italy in the late 1990s. Sequence analysis of one of these isolates was published in 2002 (NCBI-BLAST, ID AJ457137). However, studies have shown that this Italian-02 strain is widely distributed in Europe, and excluding IBV variant 4 / 91, it has become one of the most prevalent genotypes in the UK, Spain, France, and the Netherlands.

[0070] Since 1996, a new infectious bronchitis virus (IBV) genotype, known as Q1, has been prevalent in China and was first reported in Italy in 2011. Q1 is associated with increased mortality, kidney damage, and proventriculitis.

[0071] In addition, strains D274, B1648 / D8880, D1466, V1397 and Arkansas have been identified in Europe.

[0072] Where any IBV strain can be obtained is within the general knowledge of those skilled in the art. IBV strains can be commercially purchased, obtained from scientific research institutions, or their genomes can be synthesized as complementary DNA, since the IBV strain has been sequenced and the sequence is publicly available and therefore readily available. Furthermore, IBV strains can be isolated from the wild. Methods for isolating and characterizing IBV strains are well known to those skilled in the art. Valter Leonardo de Quadros 2011 (Dissertation, Das Bronchitis Virus(IBV):MolekularbiologischeUntersuchungen zur Diagnostik und zum Vorkommen sowie zur desGenotyps IBV QX in spezifisch pathogenfreien(SPF)Broilern, Freie Berlin), Worthington et al. 2009 (Avian Pathology 37(3), 247-257), Liu et al. 2009 (VirusGenes 38:56-65), Dolz et al. 2006 (Avian Pathology 35(2):77-85), Farsang et al. 2002 (Avian Pathology 31:229-236) and Feng et al. 2014 (Virus Genes 49:292-303) described how to isolate and distinguish different IBV strains.

[0073] IBV strains are typically distinguished by the coding sequence of the S1 subunit of the spike protein (Valastro et al. 2016. Infect Genet Evol. 39:349-364), but can also be distinguished by their complete nucleotide sequence or the sequence of specific proteins, such as the spike protein, nucleocapsid protein, envelope (E) protein, or membrane (M) glycoprotein. Because the spike protein determines the host tropism and antigenicity of IBV, IBV genotypes are classified by the coding sequence of the spike protein subunit 1. Alternatively, IBV strains can be distinguished by their serotype. Serotype classification involves serological assays of the virus, which involve serotype-specific antibodies.

[0074] In another specific aspect of the IBV spike protein or fragment thereof according to the invention, the spike protein or fragment thereof is derived from IBV having a genotype or serotype or strain selected from the following list: Arkansas (e.g., Arkansas 99), Brazil (e.g., BR-1, BR-2, 23 / 2013, IBV / Brasil / 351 / 1984), California (e.g., California 1734 / 04, California 99), Connecticut, Delaware (e.g., Delaware 98), Dutch (e.g., D207, D212, D274, D3128, D3896, D8880, D1466), Florida, Georgia (e.g., Georgia GA-07, GA-08, GA-12, GA-13), Gray, Holte, Iowa (e.g., Iowa 97 and Iowa 69), Italy 02, JMK, LDT3, Maine (e.g., Maine 209), Massachusetts (e.g., H52, H120, excluding M41), Pennsylvania (e.g., Pennsylvania 1220 / 98, Pennsylvania Wolg / 98), PL84084, Qu (e.g., Qu-mv), QX (e.g., GB341 / 96), Q1, SE 17, Variant 2 (e.g., IS / 1494 / 06, IBV / Ck / EG / CU / 4 / 2014, gammaCoV / Ck / Poland / G052 / 2016) and 4 / 91 (793B, CR88).

[0075] In another specific aspect of the IBV spike protein or fragment thereof according to the invention, the spike protein or fragment thereof is not derived from the Beaudette genotype or serotype.

[0076] In another specific aspect of the IBV spike protein or fragment thereof according to the invention, the spike protein or fragment thereof is not derived from the IBV strain M41.

[0077] In another specific aspect of the IBV spike protein or fragment thereof according to the invention, the spike protein or fragment thereof is derived from IBV selected from the following list of genotypes or serotypes or strains: Massachusetts (not M41), 4 / 91, QX, Q1, Italy O2, Arkansas, Conneticut, Georgia, LDT3, PL84084, variant 2 and Brazil.

[0078] The phrase "not M41" is equivalent to excluding M41. Therefore, the phrase "Massachusetts (not Beaudette)" means that it contains spike proteins or fragments thereof from Massachusetts strains such as H52 and H120, but excludes spike proteins or fragments thereof from M41 strains.

[0079] In another specific aspect of the IBV spike protein or a fragment thereof according to the invention, the spike protein or a fragment thereof is derived from IBV selected from the following list of genotypes or serotypes: Massachusetts (not M41), 4 / 91, QX, Q1, Arkansas, variant 2, and Brazil.

[0080] In another specific aspect of the IBV spike protein or a fragment thereof according to the invention, the spike protein or a fragment thereof is derived from IBV selected from the following list of genotypes or serotypes: Massachusetts (not M41) and 4 / 91.

[0081] In another specific aspect of the IBV spike protein or fragments thereof according to the invention, the Massachusetts strain is selected from the following list: H120, H52, Spain / 98 / 308, IBMA5-1, SD / 97 / 01 and Spain / 96 / 334.

[0082] In another specific aspect of the IBV spike protein or fragments thereof according to the invention, strain 4 / 91 is selected from the following list: Spain / 98 / 328, Spain / 92 / 35, IR-3654-VM, FR-CR88061-88, FR-85131-85, UK-1233-95, UK / 3 / 91, Spain / 00 / 336, UK / 7 / 91, pathogenic 4 / 91, attenuated 4 / 91, and IB4-91.

[0083] In another specific aspect of the IBV spike protein or fragments thereof according to the invention, the QX strain is selected from the following list: FR-L1450T-05, FR-L1450L-05, NL-L1449T-04, NL-L1449K-04, IBV / Ck / SP / 170 / 09, IBV / Ck / SP / 79 / 08, IBV / Ck / SP / 248 / 09, HBN, IBVQX, LX4, BJQ, CK / CH / LGD / 03 and GB341 / 96.

[0084] In another specific aspect of the IBV spike protein or a fragment thereof according to the invention, the Q1 strain is selected from the following list: CK / CH / LDL / 98I, CK / CH / LSD / 08-10, J2, Q1, AR08ER22, AR08BA21 and Chile-295-10.

[0085] In another specific aspect of the IBV spike protein or fragments thereof according to the invention, the Italy 02 strain is selected from the following list: Spain / 99 / 316, Italy-02, UK-L633-04, It-497-02, Spain / 05 / 866, Spain / 04 / 221, Spain / 00 / 337, Spain / 155 / 09 and Spain / 03 / 08.

[0086] In another specific aspect of the IBV spike protein or fragments thereof according to the invention, the Arkansas strains are selected from the following list: Ark99, ArkGA, ArkDPI, AL / 5364 / 00, ARKDPI11, AL / 0803 / 01, AL / 7149 / 00, ArkDPI101, AL / 1221 / 01, AL / 1793 / 01 and AL / 4614 / 98.

[0087] In another specific aspect of the IBV spike protein or fragments thereof according to the invention, variant 2 strains are selected from the following list: IS / 1494 / 06, IBV / Ck / EG / CU / 4 / 2014, gammaCoV / Ck / Poland / G052 / 2016, Eg / CLEVB-2 / IBV / 012, D1344 / 2 / 4 / 10_EG, TR8 and IB VAR2-06.

[0088] In another specific aspect of the IBV spike protein or fragments thereof according to the invention, the Brazil strain is selected from the following list: BR-1, BR-2, 23 / 2013 and IBV / Brasil / 351 / 1984.

[0089] In another specific aspect of the IBV spike protein or fragment thereof according to the invention, the spike protein or fragment thereof is derived from Massachusetts (not M41) or IBV of genotype 4 / 91 or serotype 4 / 91.

[0090] In another specific aspect of the IBV spike protein or fragment thereof according to the invention, the spike protein or fragment thereof is derived from the Massachusetts (not M41) genotype or serotype of IBV.

[0091] In another specific aspect of the IBV spike protein or a fragment thereof according to the invention, the spike protein or a fragment thereof is derived from IBV genotype 4 / 91 or serotype 4.

[0092] In another specific aspect of the IBV spike protein or a fragment thereof according to the invention, the IBV strain is H52, H120 or CR88.

[0093] In another specific aspect of the IBV spike protein or fragment thereof according to the invention, the IBV strain is H52 or H120.

[0094] In another specific aspect of the IBV spike protein or a fragment thereof according to the invention, the IBV spike protein or a fragment thereof consists of or comprises the following: amino acid sequences as shown in SEQ ID NO:2, 3, 4, 5, 6, 7, 8, or sequences having at least 80%, 85%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98%, or 99.99% sequence identity with them.

[0095] The term "identity" or "sequence identity" is known in the art and refers to the relationship between two or more polypeptide sequences or two or more polynucleotide sequences (i.e., a reference sequence and a given sequence to be compared with the reference sequence). Sequence identity is determined by comparing a given sequence with a reference sequence, as determined by matching strings of such sequences, after optimal alignment to produce the highest degree of sequence similarity. In such alignments, sequence identity is determined on a position-by-position basis; for example, if a nucleotide or amino acid residue is equivalent at a particular position, the sequence is "equivalent" at that position. The total number of such positional identities is then divided by the total number of nucleotides or residues in the reference sequence to give the % sequence identity. Sequence identity can be readily computed by known methods, including but not limited to those described in: Computational Molecular Biology, Lesk, AN (ed.), Oxford University Press, New York (1988); Biocomputing: Informatics and Genome Projects, Smith, DW (ed.), Academic Press, New York (1993); Computer Analysis of Sequence Data, Part I, Griffin, AM and Griffin, HG (ed.), Humana Press, New Jersey (1994); Sequence Analysis in Molecular Biology, von Heinge, G., Academic Press (1987); Sequence Analysis Primer, Gribskov, M. and Devereux, J. (ed.), M. Stockton Press, New York (1991); and Carillo, H. and Lipman, D., SIAM J. Applied Math., 48:1073 (1988), the teachings of which are incorporated herein by reference. A preferred method for determining sequence identity is designed to provide the maximum match between the tested sequences. The method for determining sequence identity is compiled in a publicly available computer program that determines sequence identity between given sequences.Examples of such programs include, but are not limited to, the GCG package (Devereux, J. et al., Nucleic Acids Research, 12(1):387(1984)), BLASTP, BLASTN, and FASTA (Altschul, SF et al., J.Molec.Biol., 215:403-410(1990). BLASTX programs are available from NCBI and other sources (BLAST Manual, Altschul, S. et al., NCVI NLM NIH Bethesda, MD). 20894, Altschul, SF et al., J. Molec. Biol., 215:403-410 (1990), the teachings of which are incorporated herein by reference. These procedures optimally align sequences using default gap weights to produce the highest level of sequence identity between a given sequence and a reference sequence. As an example, by using a polynucleotide having a nucleotide sequence having at least, for example, 85%, preferably 90%, or even more preferably 95% "sequence identity" with a reference nucleotide sequence, the nucleotide sequence of the given polynucleotide is expected to be identical to the reference sequence. Similarly, except that a given polynucleotide sequence may include at most 15, preferably at most 10, and even more preferably at most 5 point mutations per 100 nucleotides of the reference nucleotide sequence. In other words, in a polynucleotide having at least 85%, preferably 90%, and even more preferably 95% identity with the reference nucleotide sequence, at most 15%, preferably 10%, and even more preferably 5% of the nucleotides in the reference sequence may be deleted or substituted by another nucleotide, or at most 15%, preferably 10%, and even more preferably 5% of the total nucleotides in the reference sequence. Nucleotides may be inserted into the reference sequence. These mutations in the reference sequence may occur at the 5' or 3' end of the reference nucleotide sequence or at any position between these end positions, individually scattered among the nucleotides in the reference sequence or in one or more adjacent groups within the reference sequence. Similarly, with a polypeptide having a given amino acid sequence having at least, for example, 85%, preferably 90%, even more preferably 95% sequence identity with the reference amino acid sequence, the given amino acid sequence of the polypeptide is expected to be equivalent to the reference sequence, except that the given polypeptide sequence may include at most 15, preferably at most 10, even more preferably at most 5 amino acid changes per 100 amino acids of the reference amino acid sequence. In other words, to obtain a given polypeptide sequence having at least 85%, preferably 90%, even more preferably 95% sequence identity with the reference amino acid sequence, at most 15%, preferably at most 10%, even more preferably at most 5% of the amino acid residues in the reference sequence may be deleted or substituted by another amino acid, or at most 15%, preferably at most 10%, even more preferably at most 5% of the total number of amino acid residues in the reference sequence may be inserted into the reference sequence.These changes to the reference sequence may occur at the amino or carboxyl terminus of the reference amino acid sequence or at any position between these terminus, individually scattered among residues in the reference sequence or in one or more adjacent groups within the reference sequence. Preferably, the positions of non-equivalent residues differ due to conserved amino acid substitutions. However, conserved substitutions are not included as matching in determining sequence identity.

[0096] The terms “identity,” “sequence identity,” and “percentage of identity” are used interchangeably herein. For the purposes of this invention, it is defined herein as follows: to determine the percentage of identity between two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., a gap may be introduced into the sequence of the first amino acid sequence or nucleic acid sequence for optimal alignment with the second amino acid sequence or nucleic acid sequence). The amino acid or nucleotide residues at the corresponding amino acid or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid or nucleotide residue as the corresponding position in the second sequence, the molecules are identical at that position. The percentage of identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = number of identical positions / total number of positions (i.e., overlapping positions) x 100). Preferably, the two sequences have the same length.

[0097] Sequence comparisons can be performed across the entire length of the two sequences or across fragments of the two sequences. Typically, comparisons are performed across the full length of the two sequences. However, sequence identity can be performed over regions of, for example, twenty, fifty, one hundred, or more adjacent amino acid residues.

[0098] Those skilled in the art will recognize the fact that different computer programs can be used to determine the homology between two sequences. For example, the comparison of sequences and the determination of the percentage of identity between two sequences can be accomplished using mathematical algorithms. In a preferred embodiment, the percentage of identity between two amino acid sequences or nucleic acid sequences is determined using the Needleman and Wunsch (J. Mol. Biol. (48): 444-453 (1970)) algorithm, which has been incorporated into the GAP program in the Accelrys GCG software package (available at http: / / www.accelrys.com / products / gcg / ), using a Blosum 62 matrix or a PAM250 matrix, with gap weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6. Those skilled in the art will understand that all these different parameters will produce slightly different results, but the overall percentage of identity between the two sequences does not change significantly when different algorithms are used.

[0099] The protein or nucleic acid sequences of the present invention can be further used as "query sequences" to search public databases for, for example, to identify other family members or related sequences. Such searches can be performed using the BLASTN and BLASTP programs (version 2.0) of Altschul et al. (1990) J.Mol.Biol.215:403-10. BLAST protein searches can be performed using the BLASTP program with a score of 50 and a word length of 3 to obtain amino acid sequences homologous to the protein molecules of the present invention. For gap alignments for comparative purposes, Gapped BLAST can be used, as described in Altschul et al. (1997) Nucleic AcidsRes.25(17):3389-3402. When using the BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., BLASTP and BLASTN) can be used. See the homepage of the National Center for Biotechnology Information at http: / / www.ncbi.nlm.nih.gov / .

[0100] As used herein, the term “equivalent to the sequence of SEQ ID NO:X” should be understood in particular as equivalent to the terms “equivalent to the sequence of SEQ ID NO:X in length” or “equivalent to the sequence of SEQ ID NO:X in its full length”. In this context, “X” is any integer selected from 1 to 31 such that “SEQ ID NO:X” represents any SEQ ID NO mentioned herein.

[0101] In another specific aspect of the IBV spike protein or a fragment thereof according to the invention, the IBV spike protein or a fragment thereof consists of or comprises the following: an amino acid sequence as shown in SEQ ID NO:2, or a sequence having at least 80%, 85%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98%, or 99.99% sequence identity with it.

[0102] In another specific aspect of the IBV spike protein or a fragment thereof according to the invention, the IBV spike protein or a fragment thereof consists of or comprises the following: an amino acid sequence as shown in SEQ ID NO:3, or a sequence having at least 80%, 85%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98%, or 99.99% sequence identity with it.

[0103] In another specific aspect of the IBV spike protein or a fragment thereof according to the invention, the IBV spike protein or a fragment thereof consists of or comprises the following: an amino acid sequence as shown in SEQ ID NO:4, or a sequence having at least 80%, 85%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98%, or 99.99% sequence identity with it.

[0104] In another specific aspect of the IBV spike protein or a fragment thereof according to the invention, the IBV spike protein or a fragment thereof consists of or comprises the following: an amino acid sequence as shown in SEQ ID NO:5, or a sequence having at least 80%, 85%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98%, or 99.99% sequence identity with it.

[0105] In another specific aspect of the IBV spike protein or a fragment thereof according to the invention, the IBV spike protein or a fragment thereof consists of or comprises the following: an amino acid sequence as shown in SEQ ID NO:6, or a sequence having at least 80%, 85%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98%, or 99.99% sequence identity with it.

[0106] In another specific aspect of the IBV spike protein or a fragment thereof according to the invention, the IBV spike protein or a fragment thereof consists of or comprises the following: an amino acid sequence as shown in SEQ ID NO:7, or a sequence having at least 80%, 85%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98%, or 99.99% sequence identity with it.

[0107] In another specific aspect of the IBV spike protein or a fragment thereof according to the invention, the IBV spike protein or a fragment thereof consists of or comprises the following: an amino acid sequence as shown in SEQ ID NO:8, or a sequence having at least 80%, 85%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98%, or 99.99% sequence identity with it.

[0108] In another specific aspect of the IBV spike protein or a fragment thereof according to the invention, the IBV spike protein or a fragment thereof is selected from the following list of genotypes: GI-2 to 27, GII-1, GIII-1, GIV-1, GV-1, GVI-1.

[0109] Valastro et al. (2016, Infection, Genetics and Evolution 39; 349–364) described a phylogenetic-based classification system combined with the phylogenetic nomenclature for IBV strain assignment. Six genotypes (GI to GVI) were defined, collectively encompassing 32 distinct viral lineages.

[0110] In another specific aspect of the IBV spike protein or a fragment thereof according to the invention, the spike protein or a fragment thereof is not derived from the GI-1 genotype. The GI-1 genotype relates to the Massachusetts genotype / serotype.

[0111] In another specific aspect of the avian coronavirus spike protein or fragment thereof according to the invention, at least a portion of the S2 subunit of an avian coronavirus having limited cell tropism or tissue tropism is selected from: infectious bronchitis virus (IBV); guinea fowl coronavirus (GfCoV); and turkey coronavirus (TCoV; turkey enteritis virus and blue crown virus).

[0112] In another specific aspect of the avian coronavirus spike protein or fragment thereof according to the invention, at least a portion of the S2 subunit of an avian coronavirus having limited cell tropism or tissue tropism is derived from IBV (infectious bronchitis virus).

[0113] In another specific aspect of the IBV spike protein or fragments thereof according to the invention, at least a portion of the S2 subunit is derived from IBV selected from the following list of genotypes or serotypes or strains: Arkansas (e.g., Arkansas 99), Brazil (e.g., BR-1, BR-2, 23 / 2013, IBV / Brasil / 351 / 1984), California (e.g., California 1734 / 04, California 99), Connecticut, Delaware (e.g., Delaware 98), Dutch (e.g., D207, D212, D274, D3128, D3896, D8880, D1466), Florida, Georgia (e.g., Georgia GA-07, GA-08, GA-12, GA-13), Gray, Holte, Iowa (e.g., Iowa 97 and Iowa 69), Italy 02, JMK, LDT3, Maine (e.g., Maine 209), Massachusetts (e.g., H52, H120, excluding M41), Pennsylvania (e.g., Pennsylvania 1220 / 98, Pennsylvania Wolg / 98), PL84084, Qu (e.g., Qu-mv), QX (e.g., GB341 / 96), Q1, SE 17, variant 2 (e.g., IS / 1494 / 06, IBV / Ck / EG / CU / 4 / 2014, gammaCoV / Ck / Poland / G052 / 2016) and 4 / 91 (793B, CR88), or wherein at least a portion of the S2 subunit is not derived from IBV strain M41.

[0114] Therefore, in another specific aspect of the IBV spike protein or fragment thereof according to the invention, at least a portion of the S2 subunit is not derived from the IBV strain M41.

[0115] In another specific aspect of the IBV spike protein or fragment thereof according to the invention, at least a portion of the S2 subunit is derived from IBV selected from the following list of genotypes or serotypes or strains: Massachusetts (not M41), 4 / 91, QX, Q1, Italy O2, Arkansas, Conneticut, Georgia, LDT3, PL84084, variant 2, and Brazil.

[0116] In another specific aspect of the IBV spike protein or fragment thereof according to the invention, at least a portion of the S2 subunit is derived from IBV selected from the following list of genotypes or serotypes: Massachusetts (not M41), 4 / 91, QX, Q1, Arkansas, variant 2, and Brazil.

[0117] In another specific aspect of the IBV spike protein or fragments thereof according to the invention, the Massachusetts strain is selected from the following list: H120, H52, Spain / 98 / 308, IBMA5-1, SD / 97 / 01 and Spain / 96 / 334.

[0118] In another specific aspect of the IBV spike protein or fragments thereof according to the invention, strain 4 / 91 is selected from the following list: Spain / 98 / 328, Spain / 92 / 35, IR-3654-VM, FR-CR88061-88, FR-85131-85, UK-1233-95, UK / 3 / 91, Spain / 00 / 336, UK / 7 / 91, pathogenic 4 / 91, attenuated 4 / 91, and IB4-91.

[0119] In another specific aspect of the IBV spike protein or fragments thereof according to the invention, the QX strain is selected from the following list: FR-L1450T-05, FR-L1450L-05, NL-L1449T-04, NL-L1449K-04, IBV / Ck / SP / 170 / 09, IBV / Ck / SP / 79 / 08, IBV / Ck / SP / 248 / 09, HBN, IBVQX, LX4, BJQ, CK / CH / LGD / 03 and GB341 / 96.

[0120] In another specific aspect of the IBV spike protein or a fragment thereof according to the invention, the Q1 strain is selected from the following list: CK / CH / LDL / 98I, CK / CH / LSD / 08-10, J2, Q1, AR08ER22, AR08BA21 and Chile-295-10.

[0121] In another specific aspect of the IBV spike protein or fragments thereof according to the invention, the Italy 02 strain is selected from the following list: Spain / 99 / 316, Italy-02, UK-L633-04, It-497-02, Spain / 05 / 866, Spain / 04 / 221, Spain / 00 / 337, Spain / 155 / 09 and Spain / 03 / 08.

[0122] In another specific aspect of the IBV spike protein or fragments thereof according to the invention, the Arkansas strains are selected from the following list: Ark99, ArkGA, ArkDPI, AL / 5364 / 00, ARKDPI11, AL / 0803 / 01, AL / 7149 / 00, ArkDPI101, AL / 1221 / 01, AL / 1793 / 01 and AL / 4614 / 98.

[0123] In another specific aspect of the IBV spike protein or fragments thereof according to the invention, variant 2 strains are selected from the following list: IS / 1494 / 06, IBV / Ck / EG / CU / 4 / 2014, gammaCoV / Ck / Poland / G052 / 2016, Eg / CLEVB-2 / IBV / 012, D1344 / 2 / 4 / 10_EG, TR8 and IB VAR2-06.

[0124] In another specific aspect of the IBV spike protein or fragments thereof according to the invention, the Brazil strain is selected from the following list: BR-1, BR-2, 23 / 2013 and IBV / Brasil / 351 / 1984.

[0125] In another specific aspect of the IBV spike protein or fragment thereof according to the invention, at least a portion of the S2 subunit is derived from IBV selected from the following list of genotypes or serotypes: Massachusetts (not M41) and 4 / 91.

[0126] In another specific aspect of the IBV spike protein or fragment thereof according to the invention, at least a portion of the S2 subunit is derived from the IBV Massachusetts (not M41) genotype or serotype.

[0127] In another specific aspect of the IBV spike protein or a fragment thereof according to the invention, at least a portion of the S2 subunit is derived from IBV strains H120, H52, or CR88.

[0128] In another specific aspect of the IBV spike protein or a fragment thereof according to the invention, at least a portion of the S2 subunit is derived from IBV strains H120 or H52.

[0129] In another specific aspect of the avian coronavirus spike protein or fragments thereof according to the invention, at least a portion of the S2 subunit is an S2 subunit sequence from an avian coronavirus or IBV having limited cell tropism or tissue tropism, or at least 1, 5, 10, 15, 20, 25, 50, 100, 150, 200, 250, 300, 350, 400 or 500 adjacent amino acids of a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with it.

[0130] In another specific aspect of the avian coronavirus or IBV spike protein or fragments thereof according to the invention, at least a portion of the S2 subunit is a sequence of the S2 subunit of avian coronavirus or IBV as described herein, or at least 1, 5, 10, 15, 20, 25, 50, 100, 150, 200, 250, 300, 350, 400 or 500 adjacent amino acids of a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with it.

[0131] In another specific aspect of the IBV spike protein or a fragment thereof according to the invention, at least a portion of the S2 subunit of IBV having limited cell tropism or tissue tropism has an amino acid sequence as shown in SEQ ID NO:2, 3, 4, 5, 6, 7, 8, or at least 1, 5, 10, 15, 20, 25, 50, 100, 150, 200, 250, 300, 350, 400 or 500 adjacent amino acids of a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with it.

[0132] In another specific aspect of the avian coronavirus or IBV spike protein or fragment thereof according to the invention, coronaviruses or IBVs with limited cell tropism or tissue tropism are limited to infection and / or replication in embryonic eggs and / or primary chicken kidney cells.

[0133] In another specific aspect of the avian coronavirus or IBV spike protein or fragment thereof according to the invention, coronaviruses or IBV with limited cell tropism or tissue tropism do not infect and / or replicate in EB66 cells.

[0134] In another specific aspect of the avian coronavirus or IBV spike protein or fragment thereof according to the invention, coronaviruses or IBV with limited cell tropism or tissue tropism do not infect and / or replicate in BHK cells.

[0135] In another specific aspect of the avian coronavirus or IBV spike protein or fragment thereof according to the invention, coronaviruses or IBV with limited cell tropism or tissue tropism do not infect and / or replicate in PBS-12SF and / or HEK293T cells.

[0136] Excerpt

[0137] In another specific aspect of the avian coronavirus or IBV spike protein or a fragment thereof according to the invention, the fragment of the avian coronavirus or IBV spike protein has a length of at least 500, 750, 1000 or 1077 amino acids.

[0138] In another specific aspect of the avian coronavirus or IBV spike protein or a fragment thereof according to the invention, the fragment of the avian coronavirus or IBV spike protein has a length of at least 1,000 amino acids.

[0139] In another specific aspect of the avian coronavirus or IBV spike protein or a fragment thereof according to the invention, the fragment of the avian coronavirus or IBV spike protein has a length of at least 500, 750, 1000 or 1075 amino acids from the N-terminus.

[0140] The term "N-terminus" is well known to those skilled in the art. The N-terminus is also called the amino terminus, NH2 terminus, N-terminal end, or amine terminus. When a protein is translated from messenger RNA, it is generated from the N-terminus to the C-terminus. Therefore, the N-terminus is the starting point of the amino acid chain (protein or polypeptide) containing the amino group (-NH2).

[0141] In another specific aspect of the avian coronavirus or IBV spike protein or a fragment thereof according to the invention, the fragment of the avian coronavirus or IBV spike protein is an extracellular domain of the spike protein.

[0142] The term "extracellular domain" is well known to those skilled in the art. The spike protein comprises distinct functional parts, a signal sequence, an extracellular domain, a transmembrane domain, and an intracellular domain (from the N-terminus to the C-terminus). Therefore, after cleavage of the signal sequence, the N-terminus of the spike protein begins with an extracellular domain. The IBV spike extracellular domain is approximately 1075 amino acids long and varies by a few amino acids depending on the IBV strain.

[0143] In another specific aspect of the avian coronavirus or IBV spike protein according to the invention, the aromatic amino acid at amino acid position 865, or the mutation to an aromatic amino acid at amino acid position 865, is genetically stable. Advantageously, experimental data show that the aromatic amino acid at amino acid position 865, or the mutation to an aromatic amino acid at amino acid position 865, is genetically stable and remains stable over a period of time (through generations).

[0144] The term "genetically stable" means that the aromatic amino acid at amino acid position 865, or the mutation to an aromatic amino acid at amino acid position 865, is maintained over a period of time (through passage). Preferably, the aromatic amino acid at amino acid position 865, or the mutation to an aromatic amino acid at amino acid position 865, persists after at least 3 passages of IBV having the avian coronavirus or IBV spike protein according to the invention in cell or tissue culture, more preferably after at least 6 passages, even more preferably after at least 9 passages, even more preferably after at least 12 passages, and most preferably after 15 passages.

[0145] Nucleotide sequences and plasmids

[0146] Furthermore, the present invention provides nucleotide sequences encoding spike proteins or fragments thereof as described herein.

[0147] Furthermore, the present invention provides plasmids comprising the nucleotide sequences as described herein.

[0148] The terms "nucleic acid," "nucleic acid sequence," or "nucleotide sequence" refer to polynucleotides, including DNA molecules, RNA molecules, cDNA molecules, or derivatives. This term includes both single-stranded and double-stranded polynucleotides. The nucleic acids of this invention include isolated polynucleotides (i.e., isolated from their natural environment) and genetically modified forms. Furthermore, it includes chemically modified polynucleotides, including naturally occurring modified polynucleotides such as glycosylated or methylated polynucleotides, or artificially modified polynucleotides such as biotinylated polynucleotides. Further, the terms "nucleic acid" and "polynucleotide" are interchangeable and refer to any nucleic acid. Specifically, the terms "nucleic acid" and "polynucleotide" also include nucleic acids composed of nucleotides having nucleobases other than the five biologically present bases (adenine, guanine, thymine, cytosine, and uracil).

[0149] The term "plasmid" refers to cytoplasmic DNA that replicates within a bacterial host cell independently of the bacterial chromosome. In one particular aspect of the invention, the terms "plasmid" and / or "transfer plasmid" and / or "donor plasmid" refer to elements of recombinant DNA technologies that can be used to construct, for example, recombinant viruses or expression cassettes for insertion into viral vectors. In another particular aspect, the term "plasmid" can be used to specify plasmids that can be used for DNA vaccination purposes.

[0150] cell

[0151] Furthermore, the present invention provides cells comprising plasmids as described herein. The cells may be eukaryotic or prokaryotic cells.

[0152] Viral particles, avian coronavirus and IBV

[0153] Furthermore, the present invention provides viral particles comprising the spike protein or fragments thereof as described herein.

[0154] Furthermore, the present invention provides avian coronaviruses comprising the spike protein or fragments thereof as described herein.

[0155] Furthermore, the present invention provides IBV (Infectious Bronchitis Virus) comprising the spike protein as described herein.

[0156] In another specific aspect of the avian coronavirus or IBV according to the invention, the avian coronavirus or IBV is attenuated.

[0157] The term "attenuated" refers to a pathogen with reduced virulence compared to a wild-type isolate. In this invention, attenuated IBV is an IBV whose virulence has been reduced such that it does not cause clinical signs of IBV infection, but can induce an immune response in target animals. It can also mean that clinical signs are reduced in morbidity or severity in animals infected with attenuated IBV compared to a "control group" of animals infected with non-attenuated IBV and not given attenuated virus. In this context, the term "reduced / decreased" means a reduction of at least 10%, preferably 25%, even more preferably 50%, still more preferably 60%, even more preferably 70%, even more preferably 80%, even more preferably 90%, even more preferably 95%, and most preferably 100%, compared to a control group infected with non-attenuated IBV as defined above. Therefore, attenuated IBV strains are suitable for incorporation into immunogenic compositions containing modified live IBV.

[0158] In another specific aspect of the avian coronavirus or IBV according to the invention, the avian coronavirus or IBV is inactivated.

[0159] Any conventional inactivation method can be used for the purposes of this invention. Therefore, inactivation can be performed by chemical and / or physical treatments known to those skilled in the art. Preferred inactivation methods include the addition of a cyclized diethyleneimine (BEI), including a solution containing 2-bromoethylamine hydrobromide (BEA), said 2-bromoethylamine hydrobromide having been cyclized to a diethyleneimine (BEI). Preferred further chemical inactivating agents include, but are not limited to, Triton X-100, sodium deoxycholate, hexadecyltrimethylammonium bromide, β-propiolactone, thimerosal, phenol, and formaldehyde (formalin). However, inactivation may also include a neutralization step. Preferred neutralizing agents include, but are not limited to, sodium thiosulfate, sodium bisulfite, etc.

[0160] Preferred formalin inactivation conditions include formalin concentrations of about 0.02% (v / v)–2.0% (v / v), more preferably about 0.1% (v / v)–1.0% (v / v), even more preferably about 0.15% (v / v)–0.8% (v / v), even more preferably about 0.16% (v / v)–0.6% (v / v), and most preferably about 0.2% (v / v)–0.4% (v / v). The incubation time depends on the resistance of IBV. Generally, the inactivation process continues until IBV growth is undetectable in a suitable culture system.

[0161] Preferably, the inactivated IBV of the present invention is inactivated by formalin, and preferably at the concentration described above.

[0162] The inactivated IBV of the present invention can be incorporated into liposomes using known techniques, such as those described in Nature, 1974, 252, 252-254 or Journal of Immunology, 1978, 120, 1109-13. In another embodiment of the invention, the inactivated IBV of the present invention can be conjugated to suitable biological compounds such as polysaccharides, peptides, proteins, etc., or combinations thereof.

[0163] In another specific aspect of the avian coronavirus or IBV according to the invention, the avian coronavirus or IBV is genetically modified.

[0164] The term "genetically modified" refers to avian coronaviruses or IBV that have been mutated using a "reverse genetics" method. Preferably, the avian coronaviruses or IBV according to the present invention have been genetically modified. Reverse genetics techniques involve the preparation of synthetic recombinant viral RNA and / or cDNA. However, "reverse genetics" techniques are well known to those skilled in the art.

[0165] In another specific aspect of the avian coronavirus or IBV according to the invention, the avian coronavirus or IBV is recombinant.

[0166] As used herein, the term "recombinant" refers to an RNA genome (or RNA sequence, cDNA sequence, or protein) with any modifications that do not occur naturally in the corresponding RNA genome (or RNA sequence, cDNA sequence, or protein). For example, an RNA genome (or RNA sequence, cDNA sequence, or protein) is considered "recombinant" if it contains, for example, an insertion, deletion, inversion, relocation, or point mutation introduced artificially through human intervention. Thus, the RNA genome sequence (or RNA sequence, cDNA sequence, or protein) does not bind to all or part of the sequence (or RNA sequence, cDNA sequence, or protein) with which it binds in nature. When used in relation to viruses, the term "recombinant" means a virus produced through artificial manipulation of the viral genome. The term "recombinant virus" includes genetically modified viruses.

[0167] In another specific aspect of the avian coronavirus or IBV according to the invention, the avian coronavirus or IBV is chimeric.

[0168] The term "chimeric" refers to an avian coronavirus or IBV that contains one or more nucleotide sequences from another coronavirus or IBV. Preferably, the term refers to an IBV virus that contains one or more nucleotide sequences from another IBV strain.

[0169] In another specific aspect of the IBV according to the invention, the IBV is derived from IBV having a genotype or serotype or strain selected from the following list: Arkansas (e.g., Arkansas 99), Brazil (e.g., BR-1, BR-2, 23 / 2013, IBV / Brasil / 351 / 1984), California (e.g., California 1734 / 04, California 99), Connecticut, Delaware (e.g., Delaware 98), Dutch (e.g., D207, D212, D274, D3128, D3896, D8880, D1466), Florida, Georgia (e.g., Georgia GA-07, GA-08, GA-12, GA-13), Gray, Holte, Iowa (e.g., Iowa 97 and Iowa 69), Italy (e.g., Italy 02), JMK, LDT3, Maine (e.g., Maine 209), Massachusetts (M41, H52, H120, Beaudette), Pennsylvania (e.g., Pennsylvania 1220 / 98, Pennsylvania Wolg / 98), PL84084, Qu (e.g., Qu-mv), QX (e.g., GB341 / 96), Q1, SE 17, variant 2 (e.g., IS / 1494 / 06, IBV / Ck / EG / CU / 4 / 2014, gammaCoV / Ck / Poland / G052 / 2016), and 4 / 91 (793B, CR88). Therefore, it must be understood that IBV can be either the M41 genotype or serotype; however, the spike protein or fragments thereof do not originate from the IBV strain M41.

[0170] In another specific aspect of the IBV according to the invention, the IBV is selected from the following list of genotypes or serotypes or strains: Massachusetts, 4 / 91, QX, Q1, Italy 02, Arkansas, Conneticut, Georgia, LDT3, PL84084, variant 2 and Brazil.

[0171] In another specific aspect of the IBV according to the invention, the IBV is selected from the following list of genotypes or serotypes: Massachusetts, 4 / 91, QX, Q1, Arkansas, variant 2, and Brazil.

[0172] In another specific aspect of the IBV according to the invention, the Massachusetts strains are selected from the following list: H120, H52, Spain / 98 / 308, IBMA5-1, SD / 97 / 01, Beaudette, Spain / 96 / 334 and M41-M21883.

[0173] In another specific aspect of the IBV according to the invention, strain 4 / 91 is selected from the following list: Spain / 98 / 328, Spain / 92 / 35, IR-3654-VM, FR-CR88061-88, FR-85131-85, UK-1233-95, UK / 3 / 91, Spain / 00 / 336, UK / 7 / 91, pathogenic 4 / 91, attenuated 4 / 91, and IB4-91.

[0174] In another specific aspect of the IBV according to the invention, the QX strain is selected from the following list: FR-L1450T-05, FR-L1450L-05, NL-L1449T-04, NL-L1449K-04, IBV / Ck / SP / 170 / 09, IBV / Ck / SP / 79 / 08, IBV / Ck / SP / 248 / 09, HBN, IBVQX, LX4, BJQ, CK / CH / LGD / 03 and GB341 / 96.

[0175] In another specific aspect of the IBV according to the invention, the Q1 strain is selected from the following list: CK / CH / LDL / 98I, CK / CH / LSD / 08-10, J2, Q1, AR08ER22, AR08BA21 and Chile-295-10.

[0176] In another specific aspect of the IBV according to the invention, the Italy 02 strain is selected from the following list: Spain / 99 / 316, Italy-02, UK-L633-04, It-497-02, Spain / 05 / 866, Spain / 04 / 221, Spain / 00 / 337, Spain / 155 / 09 and Spain / 03 / 08.

[0177] In another specific aspect of the IBV according to the invention, the Arkansas strains are selected from the following list: Ark99, ArkGA, ArkDPI, AL / 5364 / 00, ARKDPI11, AL / 0803 / 01, AL / 7149 / 00, ArkDPI101, AL / 1221 / 01, AL / 1793 / 01 and AL / 4614 / 98.

[0178] In another specific aspect of the IBV according to the invention, variant 2 strains are selected from the following list: IS / 1494 / 06, IBV / Ck / EG / CU / 4 / 2014, gammaCoV / Ck / Poland / G052 / 2016, Eg / CLEVB-2 / IBV / 012, D1344 / 2 / 4 / 10_EG, TR8 and IB VAR2-06.

[0179] In another specific aspect of the IBV according to the invention, the Brazil strain is selected from the following list: BR-1, BR-2, 23 / 2013 and IBV / Brasil / 351 / 1984.

[0180] In another specific aspect of the IBV according to the invention, the spike protein or a fragment thereof is derived from Massachusetts or IBV of genotype 4 / 91 or serotype 4.

[0181] In another specific aspect of the IBV according to the invention, the spike protein or a fragment thereof is derived from the Massachusetts IBV genotype or serotype.

[0182] In another specific aspect of the IBV according to the invention, the spike protein or a fragment thereof is derived from IBV of genotype 4 / 91 or serotype 4.

[0183] In another specific aspect of the IBV according to the invention, the IBV strain is H120, H52 or CR88.

[0184] In another specific aspect of the IBV according to the invention, the IBV strain is H120 or H52.

[0185] In another specific aspect of the IBV according to the invention, the IBV has an IBV spike protein or fragment thereof consisting of or comprising the following amino acid sequences as shown in SEQ ID NO:2, 3, 4, 5, 6, 7, 8, or sequences having at least 80%, 85%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98%, or 99.99% sequence identity.

[0186] In another specific aspect of the IBV according to the invention, the IBV has extended cell tropism or tissue tropism.

[0187] In another specific aspect of the IBV according to the invention, IBV infects and / or replicates in at least one cell line or cell as described herein. Preferably, IBV infects and / or replicates in at least one cell line as described herein.

[0188] Furthermore, the present invention provides a cell comprising:

[0189] - Viral particles as described in this article, or

[0190] - As described in this article, avian coronavirus or IBV.

[0191] In another specific aspect of the cell according to the invention, the cell is a cell line or cell selected from the following list: primary chicken embryo cells, chicken embryo fibroblast cell lines, duck embryo stem cell lines, human embryo kidney cell lines, young hamster kidney cell lines, African green monkey kidney cell lines, rabbit kidney cell lines, canine kidney cell lines, chicken liver cell lines, bovine kidney cell lines, porcine kidney cell lines, and insect cell lines.

[0192] In another specific aspect of the cells according to the invention, the cells are cell lines selected from the following list: DF-1 (Douglas Foster), EB66 (duck embryo stem cell line), PBS-12, PBS-12SF (serum-free PBS-12), BHK21 (young hamster kidney), HEK 293T (human embryo kidney), Vero (Verda Reno), MA104, RK13 (rabbit kidney), LMH (leghorn male liver cancer), MDCK (Martin Darby dog ​​kidney), MDBK (Martin Darby bovine kidney), PK15 (pig kidney), PK2A (pig kidney), SF9, SF21, and SF+ (fall armyworm).

[0193] In another specific aspect of the cell according to the invention, the cell is a cell line selected from the following list: DF-1, EB66, PBS-12, PBS-12SF, BHK, HEK 293T, Vero, MA104 and RK13.

[0194] In another specific aspect of the cells according to the invention, the primary chicken embryo cells are fibroblasts or cells derived from liver or lung tissue.

[0195] Furthermore, the present invention provides an immunogenic composition comprising:

[0196] - As described in this article, or

[0197] - Viral particles as described in this article, or

[0198] - As described in this article, avian coronavirus or IBV.

[0199] Therefore, the present invention also provides an immunogenic composition comprising avian coronavirus or IBV, comprising the avian coronavirus or IBV spike protein or a fragment thereof, wherein at least a portion of the S2 subunit is derived from avian coronavirus or IBV having limited cell tropism or tissue tropism, and wherein the amino acid at position 865 is an aromatic amino acid. Further, the present invention also provides an immunogenic composition comprising avian coronavirus or IBV, comprising a recombinant avian coronavirus or IBV spike protein or a fragment thereof containing a mutation at amino acid position 865. Further, the amino acid sequence of SEQ ID NO:1 is used to determine the position number in the spike protein. Preferably, the amino acid sequence of the spike protein is compared with the amino acid sequence of SEQ ID NO:1.

[0200] Furthermore, the present invention provides a vaccine comprising:

[0201] - As described in this article, or

[0202] - Viral particles as described in this article, or

[0203] - As described in this article, coronavirus or IBV.

[0204] Furthermore, the present invention provides a modified live vaccine with extended cell tropism or tissue tropism, comprising:

[0205] - As described in this article, or

[0206] - Viral particles as described in this article, or

[0207] - As described in this article, coronavirus or IBV.

[0208] The term "immunogenic composition" refers to a composition comprising at least one antigen that elicits an immune response in a host to which the immunogenic composition is administered. Such an immune response may be a cellular and / or antibody-mediated immune response to the immunogenic composition of the present invention. Preferably, the immunogenic composition induces an immune response, and more preferably, confers protective immunity against one or more clinical signs of IBV infection. The host is also described as an "object." Preferably, any host or object described or mentioned herein is poultry or avian birds.

[0209] Typically, an "immune response" includes, but is not limited to, one or more of the following effects: the generation or activation of antibodies specifically targeting one or more antigens included in the immunogenic composition of the present invention, B cells, helper T cells, suppressor T cells and / or cytotoxic T cells and / or γ-δ T cells. Preferably, the host will exhibit a protective immune response or a therapeutic response.

[0210] A “protective immune response” or “protective immunity” will be demonstrated by the following: a reduction or absence of clinical signs typically exhibited by the infected host, faster recovery time and / or a decrease in the duration of infectivity in the infected host’s tissues or body fluids or excretions or a decrease in pathogen titer.

[0211] An immunogenic composition is described as a "vaccine" in cases where the host exhibits a protective immune response such that resistance to a new infection would be enhanced and / or the clinical severity of the disease would be reduced.

[0212] The terms “modified live” and “attenuated” are used interchangeably in this document.

[0213] In another specific aspect of the immunogenic composition or vaccine according to the invention, the immunogenic composition or vaccine comprises a pharmaceutically acceptable carrier.

[0214] The term "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, stabilizers, diluents, preservatives, antimicrobial and antifungal agents, isotonic agents, adsorption delay agents, adjuvants, immunostimulants, and combinations thereof.

[0215] "Diluents" can include water, saline, dextrose, ethanol, glycerol, etc. Isotonic agents can include, in particular, sodium chloride, dextrose, mannitol, sorbitol, and lactose. Stabilizers include, in particular, alkali metal salts of albumin and ethylenediaminetetraacetic acid.

[0216] In another specific aspect of the immunogenic composition or vaccine according to the invention, the pharmaceutically acceptable carrier is phosphate-buffered saline.

[0217] Preferably, the immunogenic composition further comprises a sucrose gelatin stabilizer.

[0218] Preferably, the pharmaceutically acceptable carrier is chitosan.

[0219] Chitosan is a naturally occurring deacetylated polysaccharide derived from chitin in crustaceans (e.g., shrimp, crab), insects, and other invertebrates. Recently, Rauw et al. (2009, Vet Immunol Immunop 134:249–258) demonstrated that chitosan enhances the cellular immune response to live Newcastle disease vaccines and promotes their protective effect. Furthermore, Wang et al. (2012, ArchVirol (2012) 157:1451–1461) showed results revealing the potential of chitosan as an adjuvant in live attenuated influenza vaccines.

[0220] Preferably, the immunogenic composition may further comprise one or more other immunomodulators, such as interleukins, interferons, or other cytokines. The amount and concentration of adjuvants and additives that may be used in the context of this invention can be readily determined by those skilled in the art.

[0221] In some aspects, the immunogenic compositions of the present invention contain adjuvants. As used herein, "adjuvant" may include aluminum hydroxide and aluminum phosphate, saponins such as QuilA, QS-21 (Cambridge Biotech Inc., Cambridge MA), GPI-0100 (Galenica Pharmaceuticals, Inc., Birmingham, AL), water-in-oil emulsions, oil-in-water emulsions, and water-in-oil-in-water emulsions. Emulsions may be particularly based on light liquid paraffin oils (European pharmacopoeia); isoprene-like oils, such as squalane or squalene; oils resulting from oligomerization reactions of olefins, particularly isobutylene or decene; esters of acids or alcohols containing linear alkyl groups, more particularly vegetable oils, ethyl oleate, propylene glycol di(caprylate / caprylate), glyceryl tri(caprylate / caprylate), or propylene glycol dioleate; esters of branched fatty acids or alcohols, particularly isostearates. Oils are used in combination with emulsifiers to form emulsions. Emulsifiers are preferably nonionic surfactants, particularly esters of sorbitol, dimannitol (e.g., mannitol oleate), ethylene glycol, polyglycerol, propylene glycol, and oleic acid, isostearic acid, castor oil, or hydroxystearic acid, optionally ethoxylated, and polyoxypropylene-polyoxyethylene copolymer blocks, particularly Pluronic products, especially L121. See Hunter et al., The Theory and Practical Application of Adjuvants (ed. Stewart-Tull, DES), John Wiley and Sons, NY, pp. 51–94 (1995), and Todd et al., Vaccine 15:564–570 (1997). Exemplary adjuvants are the SPT emulsion described on page 147 of “Vaccine Design, The Subunit and Adjuvant Approach”, edited by M. Powell and M. Newman, Plenum Press, 1995, and the emulsion MF59 described on page 183 of the same book.

[0222] A further example of an adjuvant is a compound selected from polymers of acrylic acid or methacrylic acid, as well as copolymers of maleic anhydride and alkenyl derivatives. Advantageous adjuvant compounds are polymers of acrylic acid or methacrylic acid, particularly those crosslinked with polyolefin ethers of sugars or polyols. These compounds are known by the term carbomer (Phameuropa Vol. 8, No. 2, June 1996). Those skilled in the art may also refer to U.S. Patent No. 2,909,462, which describes such acrylic polymers crosslinked with polyhydroxylated compounds having at least three hydroxyl groups, preferably no more than eight hydroxyl groups, wherein the hydrogen atoms of the at least three hydroxyl groups are replaced by unsaturated aliphatic groups having at least two carbon atoms. Preferred groups are those containing two to four carbon atoms, such as vinyl, allyl, and other olefinically unsaturated groups. The unsaturated group itself may contain other substituents, such as methyl. Products sold under the name Carbomer (BF Goodrich, Ohio, USA) are particularly suitable. They are crosslinked with allyl sucrose or allyl pentaerythritol. Among these, carbomer 974P, 934P, and 971P can be mentioned. The use of carbomer 971P is most preferred. Among the copolymers of maleic anhydride and alkenyl derivatives is copolymer EMA (Monsanto), which is a copolymer of maleic anhydride and ethylene. The solubility of these polymers in water results in an acidic solution preferably neutralized to physiological pH, in order to provide an adjuvant solution into which the immunogenic composition, immunological composition, or vaccine composition itself will be incorporated.

[0223] Further suitable adjuvants include, but are not limited to, the RIBI adjuvant system (Ribi Inc.), block copolymers (CytRx, Atlanta GA), SAF-M (Chiron, Emeryville CA), monophospholipid A, avridin lipid-amine adjuvant, heat-labile enterotoxins from Escherichia coli (recombinant or otherwise), cholera toxin, IMS 1314 or muramyl dipeptide, or naturally occurring or recombinant cytokines or their analogues, or stimulants of endogenous cytokine release, etc.

[0224] The adjuvant is expected to be added in an amount of about 100 μg to about 10 mg per dose, preferably about 100 μg to about 10 mg per dose, more preferably about 500 μg to about 5 mg per dose, even more preferably about 750 μg to about 2.5 mg per dose, and most preferably about 1 mg per dose. Alternatively, the adjuvant may be at a concentration of about 0.01% to 50% by volume of the final product, preferably about 2% to 30%, more preferably about 5% to 25%, even more preferably about 7% to 22%, and most preferably 10% to 20%.

[0225] In another specific aspect of the immunogenic composition or vaccine according to the invention, the immunogenic composition or vaccine effectively treats and / or prevents clinical signs caused by IBV in subjects in need. The terms "treatment and / or prevention," "clinical signs," and "need" have been defined elsewhere.

[0226] In another specific aspect of the immunogenic composition or vaccine according to the invention, the immunogenic composition or vaccine is formulated for single-dose administration.

[0227] The volume of a single dose has been defined elsewhere in this document.

[0228] Furthermore, it has been shown that a dose of the immunogenic composition of the present invention is effective following such a single-dose administration of such an immunogenic composition or vaccine.

[0229] In another specific aspect of the immunogenic composition or vaccine according to the invention, the immunogenic composition or vaccine is administered subcutaneously, intramuscularly, orally, intraovarianly, via spray, via drinking water, or by eye drops.

[0230] In another specific aspect of the immunogenic composition or vaccine according to the invention, the immunogenic composition or vaccine comprises 1 to 10 log... 10 EID50 / IBV dosage.

[0231] In another specific aspect of the immunogenic composition or vaccine according to the invention, the immunogenic composition or vaccine comprises 2 to 5 log... 10 EID50 / IBV dosage.

[0232] In another specific aspect of the immunogenic composition or vaccine according to the invention, the immunogenic composition or vaccine comprises 2 to 4 log... 10 EID50 / IBV dosage.

[0233] Methods for manufacturing, cultivation, and modification

[0234] Furthermore, the present invention provides a method for altering the cell tropism or tissue tropism of avian coronaviruses, comprising using avian coronavirus spike protein or fragments thereof as described herein.

[0235] Furthermore, the present invention provides a method for extending the cell tropism or tissue tropism of avian coronaviruses, which includes using avian coronavirus spike proteins or fragments thereof as described herein.

[0236] Furthermore, the present invention provides a method for producing or manufacturing avian coronaviruses with extended cell tropism or tissue tropism, comprising using avian coronavirus spike protein or fragments thereof as described herein.

[0237] Furthermore, the present invention provides a method for culturing avian coronaviruses in cell or tissue culture, comprising using avian coronavirus spike protein or fragments thereof as described herein.

[0238] Furthermore, the present invention provides a method for modifying avian coronaviruses, comprising modifying amino acid position 865 in the spike protein of the avian coronavirus.

[0239] Furthermore, the present invention provides a method for mutating amino acid position 865 in the spike protein of avian coronavirus, comprising:

[0240] a) Provide the nucleotide or protein sequence of the avian coronavirus spike protein.

[0241] b) Identify position 865 in the spike protein by comparison with a reference sequence.

[0242] c) Mutate position 865 of the spike protein from step b) to an aromatic amino acid.

[0243] d) Obtain the mutated spike protein from step c).

[0244] Furthermore, the present invention provides a method for mutating amino acid position 865 in the spike protein of avian coronavirus, comprising:

[0245] a) Providing avian coronavirus,

[0246] b) Identification of position 865 in the spike protein by comparison with a reference sequence.

[0247] c) Mutate position 865 of the spike protein from step b) to an aromatic amino acid.

[0248] d) Obtain the mutated avian coronavirus of step c).

[0249] The term “obtain” encompasses the harvesting, isolation, purification, and / or formulation (e.g., finishing, inactivation, and / or blending) of the spike protein or fragments thereof. The term “harvest” refers to the collection or recovery of the avian coronavirus or IBV with the modified spike protein from transfected or infected cells or cell lines. Any conventional method known in the art, such as any isolation method, can be used. Methods well known in the art include centrifugation or filtration, for example, using a semi-permeable membrane with a specific pore size. The term “isolation” includes the isolation step of the avian coronavirus or IBV with the modified spike protein. Methods for isolation from transfected or infected cells or cell lines are known to those skilled in the art. These methods include physical and / or chemical methods, including but not limited to freeze-thaw cycles, sonication, etc. Methods for “purification” of the avian coronavirus or IBV with the modified spike protein from isolates are known to those skilled in the art, such as those described in *Protein purification methods—a practical approach* (edited by ELV Harris and S. Angel, IRL Press at Oxford University Press). These methods include, but are not limited to, separation by centrifugation and / or filtration, precipitation, size exclusion (gel filtration) chromatography, affinity chromatography, metal chelation chromatography, ion exchange chromatography, covalent chromatography, hydrophobic interaction chromatography, etc. The carrier can be obtained in a purified pure form, or without or substantially without other cell material or culture medium. After the separation and / or purification, the antigen exhibits a purity of at least 80%, preferably 80%-90%, more preferably 90%-97%, most preferably more than 97%, up to an absolutely pure form free from any contamination.

[0250] According to a further aspect, as used herein, “obtaining” may also include further refining steps as part of the final preparation process, such as the addition of buffer, inactivation, neutralization, etc.

[0251] In another specific aspect of the method according to the invention, the spike protein or a fragment thereof has an aromatic amino acid at amino acid position 865.

[0252] In another specific aspect of the method according to the invention, an aromatic amino acid at position 865 is introduced by mutation.

[0253] In another specific aspect of the method according to the invention, the mutation is an amino acid substitution, deletion, or insertion.

[0254] In another specific aspect of the method according to the invention, the amino acid at amino acid position 865 is mutated to an aromatic amino acid; or the polar amino acid at amino acid position 865 is mutated to an aromatic amino acid; or the glutamine at amino acid position 865 is mutated to histidine.

[0255] In another specific aspect of the method according to the invention, avian coronavirus is IBV as described herein.

[0256] Therefore, the present invention provides a method for altering the cell tropism or tissue tropism of IBV, comprising using avian coronavirus spike protein or fragments thereof as described herein.

[0257] Therefore, the present invention provides a method for extending the cell tropism or tissue tropism of IBV, which includes using the IBV spike protein or a fragment thereof as described herein.

[0258] Therefore, the present invention provides a method for producing or manufacturing IBV with extended cell tropism or tissue tropism, comprising using IBV spike protein or fragments thereof as described herein.

[0259] Therefore, the present invention provides a method for culturing IBV in cell or tissue culture, comprising using the IBV spike protein or a fragment thereof as described herein.

[0260] Therefore, the present invention provides a method for modifying IBV, which includes modifying amino acid position 865 in the spike protein of the IBV.

[0261] Therefore, the present invention provides a method for mutating amino acid position 865 in the IBV spike protein, comprising:

[0262] a) Provide the IBV spike nucleotide or protein sequence.

[0263] b) Identify position 865 in the spike protein by comparison with a reference sequence.

[0264] c) Mutate position 865 of the spike protein from step b) to an aromatic amino acid.

[0265] d) Obtain the mutated spike protein from step c).

[0266] Therefore, the present invention provides a method for mutating amino acid position 865 in the IBV spike protein, comprising:

[0267] a) Provide IBV,

[0268] b) Identify position 865 in the spike protein by comparison with a reference sequence.

[0269] c) Mutate position 865 of the spike protein from step b) to an aromatic amino acid.

[0270] d) Obtain the mutated IBV from step c).

[0271] In another specific aspect of the method according to the invention, the coronavirus spike protein is the IBV (infectious bronchitis virus) spike protein as described herein.

[0272] In another specific aspect of the method according to the invention, the aromatic amino acid at amino acid position 865 or the mutation at amino acid position 865 results in extended cell tropism or tissue tropism.

[0273] In another specific aspect of the method according to the invention, avian coronavirus or IBV is infected and / or replicated in at least one cell line or cell as described herein.

[0274] In another specific aspect of the method according to the invention, as described herein, the numbering of amino acid position 865 is completed.

[0275] Reagent test kit

[0276] When necessary, the composition may be contained in a packaging or dispenser device, which may contain one or more unit dosage forms containing the active ingredient. The packaging may, for example, comprise metal or plastic foil, such as blister packs. The packaging or dispenser device may be accompanied by instructions for use, preferably for the intended recipient, particularly poultry. Associated with such containers may be a notification in the form prescribed by a government agency regulating the manufacture, use, or sale of the drug or biological product, reflecting approval for use by the manufacturing, using, or selling agency.

[0277] This invention provides a kit comprising viral particles, avian coronavirus, IBV, immunogenic compositions, or vaccines as described herein.

[0278] In one particular aspect of the kit according to the invention, the kit further comprises an instruction letter for treating and / or preventing poultry diseases, or an instruction letter for treating and / or preventing poultry diseases, or an instruction letter for treating and / or preventing IB.

[0279] In one particular aspect of the kit according to the invention, the kit further includes a dispenser capable of administering the vaccine to the animal.

[0280] Treatment

[0281] Furthermore, the present invention provides a method for immunizing a subject, comprising administering to such a subject an immunogenic composition as described herein.

[0282] The term "immunity" refers to active immunization, which involves applying an immunogenic composition to a subject to be immunized, thereby evoking an immune response against an antigen included in such an immunogenic composition.

[0283] Preferably, the immunization results in a reduction in the incidence of a specific avian coronavirus or IBV infection in the flock, or a reduction in the severity of clinical signs caused by or associated with a specific avian coronavirus or IBV infection.

[0284] Furthermore, the subjects in need are immunized with the immunogenic composition provided herein, resulting in protection against infection by avian coronavirus or IBV. Even more preferably, the immunization results in an effective and durable immune response against IBV infection. It should be understood that the time period will last for more than one month, preferably more than two months, preferably more than three months, more preferably more than four months, more preferably more than five months, and more preferably more than six months. It should be understood that immunization may not be effective in all immunized subjects. However, this term requires that a significant proportion of the subjects in the flock be effectively immunized.

[0285] Preferably, in this context, a group of subjects is envisioned who would typically (i.e., without immunization) develop clinical signs usually caused by or associated with avian coronavirus or IBV infection. It can be easily determined by those skilled in the art whether the subjects in said group have been effectively immunized. Preferably, immunization should be effective if, compared to subjects who were not immunized or immunized with an immunogenic composition available prior to the invention but subsequently infected with a specific avian coronavirus or IBV, clinical signs in at least 33%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, more preferably at least 95%, and most preferably 100% of the subjects in a given group are reduced in morbidity or severity by at least 10%, more preferably at least 20%, more preferably at least 30%, even more preferably at least 40%, more preferably at least 50%, even more preferably at least 60%, more preferably at least 70%, even more preferably at least 80%, more preferably at least 90%, more preferably at least 95%, and most preferably 100%.

[0286] Furthermore, the present invention provides a method for treating or preventing clinical signs caused by IBV in subjects in need, the method comprising administering to the subject a therapeutically effective amount of an immunogenic composition or vaccine as described herein.

[0287] As illustrated in the examples, the immunogenic compositions or vaccines provided herein have been proven effective in treating or preventing clinical signs induced by IBV in subjects. Therefore, experimental data show that modifying the amino acid at position 865 to an aromatic amino acid has no effect on vaccine efficacy.

[0288] The term "treatment or prevention" refers to a reduction in the incidence of a specific IBV infection in a flock of birds, or a lessening of the severity of clinical signs caused by or associated with a specific IBV infection. Therefore, the term "treatment or prevention" also refers to, compared to a group of subjects whose subjects have received an effective amount of an immunogenic composition as provided herein, a reduction in the number of subjects infected with a specific IBV (= a reduction in the incidence of specific IBV infection), or a lessening of the severity of clinical signs usually associated with or caused by IBV infection, or a reduction in viral shedding following infection with a specific IBV, or prevention or reduction of egg production decline in laying hens infected with a specific IBV.

[0289] "Treatment or prevention" generally refers to the administration of an effective amount of the immunogenic composition of the present invention to a subject or group of subjects who need or can benefit from such treatment / prevention. The term "treatment" means the administration of an effective amount of the immunogenic composition once at least some of the subjects or group of subjects have been infected with this type of IBV, and wherein such subjects have exhibited some clinical signs caused by or related to this type of IBV infection. The term "prevention" means administration to a subject prior to any IBV infection, or in the case where at least none of the subjects or group of subjects have exhibited any clinical signs caused by or related to this type of IBV infection. The terms "prophylaxis" and "preventing" are used interchangeably in this application.

[0290] As used herein, the term "effective amount" refers to, but is not limited to, the amount of antigen that elicits or is capable of eliciting an immune response in an object. Such effective amounts can reduce the incidence of a specific IBV infection in a flock of birds or alleviate the severity of the clinical signs of a specific IBV infection.

[0291] Preferably, compared with untreated subjects or subjects treated with immunogenic compositions available prior to the present invention but subsequently infected with a specific IBV, clinical signs are reduced in morbidity or severity by at least 10%, more preferably at least 20%, even more preferably at least 30%, even more preferably at least 40%, even more preferably at least 50%, even more preferably at least 60%, even more preferably at least 70%, even more preferably at least 80%, even more preferably at least 90%, even more preferably at least 95%, and most preferably 100%.

[0292] As used herein, the term "clinical signs" refers to the signs of infection in an object derived from IBV. The clinical signs of infection depend on the chosen pathogen. Examples of such clinical signs include, but are not limited to, respiratory distress, nephritis, salpingitis, abnormal egg production, ruffled feathers, depression, slowed growth rate, and decreased appetite. Signs of respiratory distress include respiratory signs such as wheezing, coughing, sneezing, tracheal rales, nasal and ocular discharge, tracheal damage, and ciliary arrest in the trachea. Signs of nephritis include kidney damage and watery diarrhea. Signs of abnormal egg production include decreased egg production, smaller eggs, poor eggshell quality, reduced internal egg quality, eggs with thin albumen, and ciliary arrest in the oviduct. However, clinical signs also include, but are not limited to, clinical signs that can be directly observed in living animals. Examples of clinical signs that can be directly observed in living animals include nasal and ocular discharge, cough, wheezing, sneezing, tracheal rales, ruffled feathers, conjunctivitis, weight loss, slowed growth rate, decreased appetite, dehydration, watery diarrhea, lameness, lethargy, emaciation, and weakness, among others.

[0293] Preferably, compared with untreated subjects or subjects treated with immunogenic compositions available prior to this invention but subsequently infected with a specific IBV, the clinical signs of reduced morbidity or severity in treated subjects include reduced ciliary arrest, reduced rales, reduced egg production, reduced kidney damage, reduced watery diarrhea, reduced weight loss, lower viral load, reduced viral shedding, or combinations thereof.

[0294] As used herein, the terms “need” or “required” mean that the administration / treatment is associated with the enhancement or improvement of health or clinical signs, or any other positive medical effect on the health of the subject receiving the immunogenic composition according to the invention.

[0295] Furthermore, the present invention provides a method for reducing ciliary stagnation in a desired subject compared to a non-immunized control group of the same species, the method comprising administering to the subject a therapeutically effective amount of an immunogenic composition or vaccine as described herein.

[0296] As shown in the examples, the immunogenic compositions or vaccines provided herein have been proven to effectively reduce ciliary stagnation.

[0297] The term "ciliary stagnation" refers to a reduction in ciliary movement in the trachea. Therefore, ciliary stagnation can be determined by examining the lining of the tracheal rings in terms of ciliary movement. How to determine ciliary movement in the trachea is within the general knowledge of those skilled in the art.

[0298] Preferably, compared with a non-immune control group of the same species, the movement of cilia does not decrease from day 10 after IBV attack or infection, more preferably from day 5 after attack or infection, even more preferably from day 4 after attack or infection, even more preferably from day 3 after attack or infection, and most preferably from day 1 or day 2 after attack or infection.

[0299] The term "reduction in ciliary stasis" means that, compared with a non-immunized control group of the same species, ciliary stasis is reduced by at least 10%, preferably at least 20%, more preferably at least 30%, even more preferably at least 40%, even more preferably at least 50%, even more preferably at least 60%, even more preferably at least 70%, even more preferably at least 80%, even more preferably at least 90%, even more preferably at least 95%, and most preferably 100%. How to measure the reduction in ciliary stasis is within the general knowledge of those skilled in the art.

[0300] Furthermore, the present invention provides an immunogenic composition or vaccine as described herein, in a method for immunizing a subject, the method comprising administering a therapeutically effective amount of the immunogenic composition or vaccine to the subject.

[0301] Furthermore, the present invention provides an immunogenic composition or vaccine as described herein, in a method for treating or preventing clinical signs caused by IBV in a subject of need, the method comprising administering a therapeutically effective amount of the immunogenic composition or vaccine to the subject.

[0302] Furthermore, the present invention provides an immunogenic composition or vaccine as described herein for use in a method of reducing ciliary stagnation in a desired subject compared to a non-immunized control group of the same species, the method comprising administering a therapeutically effective amount of the immunogenic composition or vaccine to the subject.

[0303] In one particular aspect of the method or use according to the invention, the object is poultry.

[0304] The term "poultry" is well known to those skilled in the art. The term "poultry" includes all birds, including poultry.

[0305] In one particular aspect of the method or use according to the invention, the object is poultry.

[0306] The term "poultry" is well known to those skilled in the art. The term "poultry" includes chickens, turkeys, quails, pheasants, guinea fowl, geese, and ducks. Further, the term "chicken" includes broilers, laying hens, and breeding stock used for both, also referred to as breeder chickens.

[0307] In one particular aspect of the method or use according to the invention, the object is selected from chickens, turkeys, quails, or pheasants.

[0308] In one particular aspect of the method or use according to the invention, the object is a chicken.

[0309] In one particular aspect of the method or use according to the invention, the immunogenic composition or vaccine is administered once.

[0310] It should be understood that a single dose is administered only once. As shown in the examples, the immunogenic compositions provided herein have proven effective after a single dose is administered to a subject in need.

[0311] The dose-volume ratio per poultry depends on the route of vaccination and the age of the poultry.

[0312] Typically, eye drop vaccines are administered at any age in volumes of 1 to 100 μl per dose. Preferably, a single dose of the eye drop vaccine has a total volume of about 5 μl to 70 μl, and more preferably about 20 μl to 50 μl, wherein single doses of 20 μl, 25 μl, 30 μl, 35 μl, 40 μl, 45 μl, or 50 μl are preferred. Most preferably, a single dose of the eye drop vaccine has a total volume of about 30 μl to 50 μl, wherein single doses of 30 μl, 35 μl, 40 μl, 45 μl, or 50 μl are preferred.

[0313] For day-old poultry, the spray vaccine may contain a dose in volumes of 25 to 1000 μl. Preferably, a single dose of the spray vaccine has a total volume of about 50 μl to 5000 μl, more preferably about 75 μl to 2000 μl, more preferably about 100 μl to 1000 μl, even more preferably about 200 μl to 900 μl, even more preferably about 300 μl to 800 μl, and even more preferably about 400 μl to 700 μl, wherein individual doses of 400 μl, 425 μl, 450 μl, 475 μl, 500 μl, 525 μl, 550 μl, 575 μl, 600 μl, 625 μl, 650 μl, 675 μl, or 700 μl are preferred. Most preferably, a single dose has a total volume of 400 μl, 450 μl, 500 μl, 550 μl, 600 μl, 650 μl or 700 μl.

[0314] Vaccines for intraovarian vaccination may contain a dose of 50 to 100 μl, preferably 50 μl. Preferably, a single dose of the intraovarian vaccine has a total volume of about 10 μl to 250 μl, more preferably about 15 μl to 200 μl, even more preferably about 20 μl to 150 μl, even more preferably about 30 μl to 100 μl, even more preferably about 30 μl to 75 μl, and wherein individual doses of 30 μl, 35 μl, 40 μl, 45 μl, 50 μl, 55 μl, 60 μl, 65 μl, 70 μl, or 75 μl are preferred. Most preferably, a single dose has a total volume of 40 μl, 45 μl, 50 μl, 55 μl, or 60 μl.

[0315] A single dose of a vaccine for intramuscular or subcutaneous administration or a drinking water vaccine may contain a volume of 30 μl to 1000 μl. Preferably, a single dose has a total volume of about 30 μl to 1000 μl, more preferably about 50 μl to 500 μl, even more preferably about 75 μl to 250 μl, and even more preferably about 100 μl to 200 μl, wherein single doses of 100 μl, 110 μl, 120 μl, 125 μl, 130 μl, 135 μl, 140 μl, 145 μl, 150 μl, 160 μl, 170 μl, 175 μl, 180 μl, 190 μl, 155 μl, or 200 μl are most preferred.

[0316] In one particular aspect of the method or use according to the invention, the immunogenic composition or vaccine is administered in two or more doses.

[0317] However, the immunogenic composition may be administered in two or more doses, wherein the first dose is administered before the second (enhanced) dose is administered.

[0318] In a preferred aspect of the two-dose regimen, the first and second doses of the immunogenic composition are administered in the same amount. Preferably, each dose is the preferred amount specified above. In addition to the first and second dose regimen, an alternative embodiment includes further subsequent doses. For example, a third, fourth, or fifth dose may be administered in these aspects. Preferably, the subsequent third, fourth, and fifth dose regimens are administered in the same amount as the first dose, wherein the time frame between the doses coincides with the time arrangement between the first and second doses mentioned above.

[0319] Preferably, the first administration of the vaccine is given within the first three weeks of age, more preferably within the first week of age, and most preferably at one day of age, by the method described below. The second administration can be given within the first 20 weeks of age, preferably within 16-18 weeks of age, and more preferably between 6-12 weeks of age. Exemplarily, the initial (first) vaccination is given at 1-10 days of age, while the second vaccination (booster) is given at 6-12 or 16-18 weeks of age using a live or inactivated vaccine. More preferably, the initial (first) vaccination is given at one day of age, while the second vaccination (booster) is given at 6-12 or 16-18 weeks of age using a live or inactivated vaccine.

[0320] When using intraovarian vaccination, the first administration is preferably performed when the embryo is 15 to 19 days old, more preferably at 17, 18, or 19 days old, and most preferably at 18 days old. The second administration can be performed within the first three weeks of life, preferably within the first 10 days of life.

[0321] In one particular aspect of the method or use according to the invention, the immunogenic composition or vaccine is administered subcutaneously, intramuscularly, orally, intraovarianly, via spray, via drinking water, or by eye drops.

[0322] Immunogenic compositions are preferably administered topically or systemically. Suitable routes of administration are conventionally used, such as oral or parenteral, including intranasal, intravenous, intradermal, percutaneous, intramuscular, intraperitoneal, subcutaneous, inhalation, intraovarian, via spray, via drinking water, or via eye drops. However, depending on the nature and mode of action of the compound, immunogenic compositions may also be administered via other routes. These other routes include intradermal, intravenous, intravascular, intraarterial, intraperitoneal, intrathecal, intratracheal, intradermal, intracardiac, intralobar, intramedullary, intrapulmonary, rectal, and vaginal administration. However, most preferably, the immunogenic composition is administered subcutaneously, intramuscularly, orally, intraovarianly, via spray, via drinking water, or via eye drops.

[0323] IBV live vaccines are preferably administered individually via eye drops, intranasal administration, intramuscular administration, or subcutaneous administration.

[0324] More preferably, large-scale application methods are used, including drinking water and aerosol spray vaccination. The use of a vaccine as an embryonic vaccine (so-called intraovarian vaccine) is also preferred, as further described below.

[0325] For example, broilers can be vaccinated at one day of age or 1-3 weeks of age, especially for broilers with high levels of MDA. Laying or breeding flocks can be initially vaccinated at 1-10 days of age and boosted with a vaccine at 7-12 or 16-18 weeks of age.

[0326] Intraocular administration

[0327] As summarized above, the present invention also provides an IBV vaccine that can be safely administered via the intraocular route and simultaneously induces a protective immune response. Intraocular administration is well known to those skilled in the art and can be performed effortlessly by those skilled in the art. Intraocular administration of the vaccine involves administering the vaccine to the avian embryo contained within the egg (for a review of intraocular vaccination, see: Ricks et al., Advances in Vet. Med. 495-515, 1999). As described in the art (Sharma; Am. J. Vet. Res. 45 1619-1623, 1984), the vaccine can be administered to any suitable egg compartment (e.g., allantoic fluid, yolk sac, amnion, air cell, or embryo). Preferably, the vaccine is administered below the shell (air cell) membrane and the allantoic membrane.

[0328] Preferably, the vaccine is injected into the embryonic eggs during the later stages of embryonic development, generally in the last quarter of the incubation period, preferably 3-4 days before hatching. Preferably, the administration is performed when the embryo is 15 to 19 days old, more preferably at 17, 18, or 19 days old, and most preferably at 18 days old. Subsequently, the vaccine-injected embryonic eggs are transferred to an incubator for hatching. The intra-egg administration process can be automated using a robotic injection procedure as described in the prior art.

[0329] Conventional vaccines used for post-hatching vaccination of poultry cannot be used for intraocular vaccination because late-stage embryos are highly susceptible to infection by most vaccine viruses examined. However, International Patent Application WO 01 / 64244 discloses that IBV vaccines can be administered intraocularly, provided they are administered at extremely low doses. Furthermore, Wakenell et al. (1986, Am. J. Vet. Res., 47 933-938) disclosed passage of IB vaccine viruses in tissue culture to render the virus non-pathogenic to embryos.

[0330] In one particular aspect of the method or use according to the invention, the immunogenic composition or vaccine is administered via eye drops.

[0331] Typically, live vaccines used for post-hatching administration contain 10 1 Up to 10 8 EID 50 Attenuated IBV titer of (50% ovarian infection dose) / dose, preferably with 10 2 Up to 10 5 EID 50 / dose of virus titer, and more preferably 10 2 Up to 10 4 EID 50 / viral titer per unit dose, and even more preferably 10 2 Up to 10 3 EID 50 / Viral titer of the dose.

[0332] Live vaccines for intraovarian administration are typically contained in a volume of 50 to 100 μl, preferably 50 μl, of 10 2 Up to 10 7 EID 50 / Embryo, preferably 10 2 Up to 10 3 EID 50 / The amount of attenuated IBV in the embryo.

[0333] Preferably, the immunogenic composition of the present invention comprises about 1 to about 10 log. 10 EID (Egg Infection Dosage) 50 The IBV of the present invention is preferably about 2 to about 8 log per ml / dose, with a preferred concentration of about 2 to about 8 log. 10 EID 50 / Dosage, preferably from about 2 to about 7 log 10 EID 50 / The amount of the dose, more preferably from about 2 to about 6 log 10 EID 50 / The amount of dose, even more preferably about 2 to about 5 log 10 EID 50 / The amount of dose, even more preferably about 2 to about 4 log 10 EID 50 The dosage is preferably about 2 to about 3 log. 10 EID 50 / Amount of dosage. More preferably, the immunogenic composition of the present invention comprises an amount of about 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5 or log. 10 EID 50 / Dosage of the IBV of the present invention.

[0334] In a particular aspect of the method or use according to the invention, the immunogenic composition or vaccine contains 1 to 10 log... 10 EID 50 / Dosage of IBV.

[0335] In a particular aspect of the method or use according to the invention, the immunogenic composition or vaccine comprises 2 to 5 log [unclear text - possibly a number or percentage]. 10 EID 50 / Dosage of IBV.

[0336] In a particular aspect of the method or use according to the invention, the immunogenic composition or vaccine comprises 2 to 4 log... 10 EID 50 / Dosage of IBV.

[0337] In one particular aspect of the method or use according to the invention, the immunogenic composition or vaccine is administered to a subject within the first week of life, within the first three days of life, within the first two days of life, or within the first day of life.

[0338] Preferably, the subjects to be immunized are 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 days old. More preferably, the subjects to be immunized are 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days old. Most preferably, the subjects to be immunized are 1, 2, 3, 4, 5, 6, or 7 days old.

[0339] However, it must be understood that poultry's immune system does require several days to develop immunity against IBV infection after vaccination in subjects a few days old. Therefore, it is preferable that subjects be immunized within the first 24 hours of age.

[0340] In one particular aspect of the method or use according to the invention, the immunogenic composition or vaccine is administered to subjects within the previous day of age. As shown in the examples, the immunogenic compositions provided herein have proven to be safe and effective when administered to 1-day-old poultry.

[0341] In a particular aspect of the method or use according to the invention, the method results in an improvement in efficacy parameters selected from the following: prevention or reduction of ciliary arrest, prevention or reduction of rales, prevention or reduction of decreased egg production, prevention or reduction of kidney damage, prevention or reduction of watery diarrhea, prevention or reduction of weight loss, lower viral load, reduced viral shedding, or a combination thereof, compared to an untreated control group of the same species.

[0342] The term “treatment and / or prevention” has been defined elsewhere, and the terms “prophylaxis” and “preventing” or “prevention” are used interchangeably in this application. Furthermore, the term “shedding” has also been defined elsewhere.

[0343] The terms “reducing,” “reduced,” “reduction,” or “lowering” mean that, compared with a non-immunized control group of the same species, the efficacy parameters (cilia arrest, rales, decreased egg production, kidney damage, watery diarrhea, weight loss, viral load, viral shedding) are reduced by at least 10%, preferably at least 20%, more preferably at least 30%, even more preferably at least 40%, even more preferably at least 50%, even more preferably at least 60%, even more preferably at least 70%, even more preferably at least 80%, even more preferably at least 90%, even more preferably at least 95%, and most preferably 100%. How to measure the improvement in efficacy parameters is within the general knowledge of those skilled in the art.

[0344] The terms “viral load” or “viral titer” are measures of the severity of an active viral infection and can be determined by methods known to those skilled in the art. The term “viral titer” is a measure of infectious units / volume of viral agent. Viral titer is an endpoint in biological procedures and is defined as the dilution to which a certain proportion of parallel tests show an effect (Reed and Muench, 1938). Determination can be based on, for example, detection of viral proteins by antibodies binding to viral proteins, and further or alternative detection by amplification methods such as RT-PCR of viral RNA. Monitoring viral particle-associated viral RNA in plasma by nucleic acid amplification methods is a widely used parameter to evaluate the status and progression of retroviral diseases and to assess the effectiveness of preventive and therapeutic interventions. Exemplarily, viral load or viral titer can be calculated by estimating the viral activity in the involved bodily fluids, such as the number of RNA copies per milliliter of plasma.

[0345] The term "ciliary arrest" is well known to those skilled in the art. The surface of the trachea is covered with specialized epithelial cells, lined with numerous, active, hair-like structures called cilia. The term "ciliary arrest" includes a reduction or loss of cilia and / or a loss or partial loss of ciliary activity. Ciliary arrest can be determined effortlessly by those skilled in the art.

[0346] The term "rales" is well known to those skilled in the art. However, the term "rales" includes tracheal rales and refers to sounds emanating from the bronchi. A person skilled in the art can readily identify rales.

[0347] The term "decline in egg production" is well known to those skilled in the art. The term "decline in egg production" includes reduced egg production.

[0348] In a particular aspect of the method or use according to the invention, treatment or prevention leads to the prevention or reduction of ciliary arrest compared to an untreated control group of the same species.

[0349] In a particular aspect of the method or use according to the invention, treatment or prevention leads to the prevention or reduction of kidney damage compared to an untreated control group of the same species.

[0350] In a particular aspect of the method or use according to the invention, treatment or prevention leads to a reduction or decrease in egg production compared to an untreated control group of the same species.

[0351] The present invention further provides viral particles, avian coronaviruses, IBV, immunogenic compositions or vaccines as described herein for therapeutic use.

[0352] The present invention further provides viral particles, avian coronaviruses, or IBV as described herein, which can be used as immunogens or vaccines.

[0353] The present invention further provides viral particles, avian coronaviruses, IBV, immunogenic compositions or vaccines as described herein, which are used as pharmaceuticals.

[0354] The present invention further provides the use of viral particles, avian coronaviruses, IBV, immunogenic compositions or vaccines as described herein for the manufacture of pharmaceuticals.

[0355] The present invention further provides the use of viral particles, avian coronaviruses, IBV, immunogenic compositions or vaccines as described herein for the treatment and / or prevention of IBV infection in subjects.

[0356] Technical solution

[0357] This article also describes the following technical solutions:

[0358] 1. An avian coronavirus spike protein or a fragment thereof comprising an aromatic amino acid at position 865, wherein the spike protein or the fragment thereof is not derived from IBV strain M41.

[0359] 2. An avian coronavirus spike protein or a fragment thereof, wherein at least a portion of the S2 subunit is derived from an avian coronavirus having limited cell tropism or tissue tropism, and wherein the amino acid at position 865 is an aromatic amino acid.

[0360] 3. A recombinant avian coronavirus spike protein or a fragment thereof, comprising a mutation at amino acid position 865.

[0361] 4. An IBV spike protein or a fragment thereof comprising an aromatic amino acid at position 865, wherein the spike protein or fragment thereof is not derived from IBV strain M41.

[0362] 5. An IBV spike protein or a fragment thereof, wherein at least a portion of the S2 subunit is derived from IBV having limited cell tropism or tissue tropism, and wherein the amino acid at position 865 is an aromatic amino acid.

[0363] 6. A recombinant IBV spike protein or a fragment thereof, comprising a mutation at amino acid position 865.

[0364] Mutation 865

[0365] 7. The avian coronavirus or IBV spike protein or fragment thereof of technical solution 1, 2, 4 or 5, wherein an aromatic amino acid at amino acid position 865 is introduced by mutation.

[0366] 8. The avian coronavirus or IBV spike protein or fragment thereof of technical solution 3, 6 or 7, wherein the mutation is an amino acid substitution, deletion or insertion.

[0367] 9. The avian coronavirus or IBV spike protein or a fragment thereof of any one of technical solutions 3 and 6 to 8, wherein the amino acid at amino acid position 865 is mutated to an aromatic amino acid; or the polar amino acid at amino acid position 865 is mutated to an aromatic amino acid; or the glutamine at amino acid position 865 is mutated to histidine.

[0368] 10. The avian coronavirus or IBV spike protein or a fragment thereof of any one of technical solutions 1, 2, 4, 5 and 7, wherein the aromatic amino acid at amino acid position 865 is histidine.

[0369] Extended cell tropism or tissue tropism

[0370] 11. The avian coronavirus or IBV spike protein or a fragment thereof of any one of technical solutions 1 to 10, wherein the aromatic amino acid at amino acid position 865 or the mutation at amino acid position 865 results in extended cell tropism or tissue tropism of the avian coronavirus or IBV.

[0371] 12. The avian coronavirus or IBV spike protein or fragment thereof of any one of technical solutions 1 to 11, wherein the avian coronavirus or IBV infects and / or replicates in at least one cell line or cell selected from the following list: primary chicken embryo cells or primary chicken fibroblasts from the lung or liver, chicken embryo fibroblast cell lines, duck embryo stem cell lines, human embryo kidney cell lines, young hamster kidney cell lines, African green monkey kidney cell lines, rabbit kidney cell lines, canine kidney cell lines, chicken liver cell lines, bovine kidney cell lines, porcine kidney cell lines, and insect cell lines.

[0372] 13. The avian coronavirus or IBV spike protein or fragment thereof of any one of technical solutions 1 to 12, wherein the avian coronavirus or IBV is infected and / or replicated in at least one cell line selected from the following list: DF-1 (Douglas Foster), EB66 (duck embryo stem cell line), PBS-12, PBS-12SF (serum-free PBS-12), BHK21 (juvenile hamster kidney), HEK293T (human embryo kidney), Vero (Verda Reno), MA104, RK13 (rabbit kidney), LMH (leghorn male liver cancer), MDCK (Martin Darby canine kidney), MDBK (Martin Darby bovine kidney), PK15 (porcine kidney), PK2A (porcine kidney), SF9, SF21, and SF+ (fall armyworm).

[0373] 14. The avian coronavirus or IBV spike protein or fragment thereof of any one of technical solutions 1 to 13, wherein the avian coronavirus or IBV infects and / or replicates in at least one cell line selected from the following list: DF-1, EB66, PBS-12, PBS-12SF, BHK, HEK 293T, Vero, MA104 and RK13.

[0374] Amino acid position 865 numbering

[0375] 15. The avian coronavirus or IBV spike protein or a fragment thereof of any one of technical solutions 1 to 14, wherein the numbering of amino acid position 865 refers to amino acid position 865 in the spike protein of IBV H52, IBV H120 or M41.

[0376] 16. The avian coronavirus or IBV spike protein or a fragment thereof of any one of technical solutions 1 to 14, wherein the number of amino acid position 865 refers to amino acid position 865 in the spike protein as exemplarily given in SEQ ID NO:1.

[0377] 17. The avian coronavirus or IBV spike protein or a fragment thereof of any one of technical solutions 1 to 14, wherein the amino acid sequence of SEQ ID NO:1 is used to determine the position number in the spike protein.

[0378] 18. The avian coronavirus or IBV spike protein or a fragment thereof of any one of technical solutions 1 to 14, wherein, in order to determine the amino acid position 865 in the spike protein, the amino acid sequence is compared with the amino acid sequence of SEQ ID NO:1.

[0379] 19. The avian coronavirus or IBV spike protein or a fragment thereof of any one of technical solutions 1 to 18, wherein the amino acid position 865 is within the S2 subunit of the spike protein.

[0380] 20. The avian coronavirus or IBV spike protein or a fragment thereof of any one of technical solutions 1 to 19, wherein the spike protein or the fragment thereof has one or more amino acids selected from the following:

[0381] -862 is alanine, and / or

[0382] -864 is alanine, and / or

[0383] -866 is valine, and / or

[0384] -867 is aspartic acid, and / or

[0385] -868 is arginine.

[0386] spike

[0387] 21. The avian coronavirus spike protein or a fragment thereof of any one of technical solutions 1 to 3 and 7 to 20, wherein the avian coronavirus spike protein or a fragment thereof is selected from: infectious bronchitis virus (IBV); guinea fowl coronavirus (GfCoV) and turkey coronavirus (TCoV; turkey enteritis virus and blue crown virus).

[0388] 22. The avian coronavirus spike protein or a fragment thereof of any one of technical solutions 1 to 3 and 7 to 21, wherein the avian coronavirus is IBV (infectious bronchitis virus).

[0389] 23. The IBV spike protein or a fragment thereof of any one of technical claims 4 to 22, wherein the spike protein or fragment thereof is derived from IBV having a genotype or serotype or strain selected from the following list: Arkansas (e.g., Arkansas 99), Brazil (e.g., BR-1, BR-2, 23 / 2013, IBV / Brasil / 351 / 1984), California (e.g., California 1734 / 04, California 99), Connecticut, Delaware (e.g., Delaware 98), Dutch (e.g., D207, D212, D274, D3128, D3896, D8880, D1466), Florida, Georgia (e.g., Georgia GA-07, GA-08, GA-12, GA-13), Gray, Holte, Iowa (e.g., Iowa 97 and Iowa 69), Italy 02, JMK, LDT3, Maine (e.g., Maine 209), Massachusetts (e.g., H52, H120, excluding M41), Pennsylvania (e.g., Pennsylvania 1220 / 98, Pennsylvania Wolg / 98), PL84084, Qu (e.g., Qu-mv), QX (e.g., GB341 / 96), Q1, SE 17, Variant 2 (e.g., IS / 1494 / 06, IBV / Ck / EG / CU / 4 / 2014, gammaCoV / Ck / Poland / G052 / 2016) and 4 / 91 (793B, CR88).

[0390] 24. The IBV spike protein or a fragment thereof of any one of technical solutions 4 to 22, wherein the spike protein or fragment thereof is not derived from the Beaudette genotype or serotype.

[0391] 25. The IBV spike protein or a fragment thereof of any one of technical solutions 4 to 24, wherein the spike protein or the fragment thereof is not derived from IBV strain M41.

[0392] 26. The IBV spike protein or a fragment thereof of any one of technical claims 4 to 25, wherein the spike protein or fragment thereof is derived from IBV selected from the following list of genotypes or serotypes or strains: Massachusetts (not M41), 4 / 91, QX, Q1, Italy 02, Arkansas, Conneticut, Georgia, LDT3, PL84084, variant 2, and Brazil.

[0393] 27. The IBV spike protein or a fragment thereof of any one of claims 4 to 26, wherein the spike protein or fragment thereof is derived from IBV selected from the following list of genotypes or serotypes: Massachusetts (not M41), 4 / 91, QX, Q1, Arkansas, variant 2, and Brazil.

[0394] 28. The IBV spike protein or a fragment thereof of any one of technical claims 4 to 27, wherein the spike protein or fragment thereof is derived from IBV selected from the following list of genotypes or serotypes: Massachusetts (not M41) and 4 / 91.

[0395] 29. The IBV spike protein or a fragment thereof of technical claim 27, wherein the Massachusetts strain is selected from the following list: H120, H52, Spain / 98 / 308, IBMA5-1, SD / 97 / 01, Beaudette and Spain / 96 / 334.

[0396] 30. The IBV spike protein or a fragment thereof of technical claim 27, wherein the 4 / 91 strain is selected from the following list: Spain / 98 / 328, Spain / 92 / 35, IR-3654-VM, FR-CR88061-88, FR-85131-85, UK-1233-95, UK / 3 / 91, Spain / 00 / 336, UK / 7 / 91, pathogenic 4 / 91, attenuated 4 / 91, and IB4-91.

[0397] 31. The IBV spike protein or a fragment thereof of technical claim 27, wherein the QX strain is selected from the following list: FR-L1450T-05, FR-L1450L-05, NL-L1449T-04, NL-L1449K-04, IBV / Ck / SP / 170 / 09, IBV / Ck / SP / 79 / 08, IBV / Ck / SP / 248 / 09, HBN, IBVQX, LX4, BJQ, CK / CH / LGD / 03 and GB341 / 96.

[0398] 32. The IBV spike protein or a fragment thereof of technical claim 27, wherein the Q1 strain is selected from the following list: CK / CH / LDL / 98I, CK / CH / LSD / 08-10, J2, Q1, AR08ER22, AR08BA21 and Chile-295-10.

[0399] 33. The IBV spike protein or a fragment thereof of technical claim 27, wherein the Arkansas strain is selected from the following list: Ark99, ArkGA, ArkDPI, AL / 5364 / 00, ARKDPI11, AL / 0803 / 01, AL / 7149 / 00, ArkDPI101, AL / 1221 / 01, AL / 1793 / 01 and AL / 4614 / 98.

[0400] 34. The IBV spike protein or a fragment thereof of technical claim 27, wherein the variant 2 strain is selected from the following list: IS / 1494 / 06, IBV / Ck / EG / CU / 4 / 2014, gammaCoV / Ck / Poland / G052 / 2016, Eg / CLEVB-2 / IBV / 012, D1344 / 2 / 4 / 10_EG, TR8 and IB VAR2-06.

[0401] 35. The IBV spike protein or a fragment thereof of technical claim 27, wherein the Brazil strain is selected from the following list: BR-1, BR-2, 23 / 2013 and IBV / Brasil / 351 / 1984.

[0402] 36. The IBV spike protein or a fragment thereof of any one of technical solutions 4 to 27, wherein the spike protein or the fragment thereof is derived from Massachusetts (not M41) or IBV of genotype 4 / 91 or serotype.

[0403] 37. The IBV spike protein or a fragment thereof of any one of technical solutions 4 to 27, wherein the IBV strain is H52, H120 or CR88.

[0404] 38. The IBV spike protein or fragment thereof of any one of technical solutions 4 to 37, wherein the IBV spike protein or fragment thereof consists of or comprises the following: amino acid sequences as shown in SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, or sequences having at least 80%, 85%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98%, or 99.99% sequence identity with them.

[0405] 39. The IBV spike protein or a fragment thereof of any one of technical solutions 4 to 38, wherein the IBV spike protein or a fragment thereof is selected from the following list of genotypes: GI-2 to 27, GII-1, GIII-1, GIV-1, GV-1, GVI-1.

[0406] 40. The IBV spike protein or a fragment thereof of any one of technical solutions 4 to 39, wherein the spike protein or the fragment thereof is not derived from the GI-1 genotype.

[0407] 41. The avian coronavirus spike protein or a fragment thereof of any one of technical claims 2 and 7 to 40, wherein at least a portion of the S2 subunit of the avian coronavirus having limited cell tropism or tissue tropism is selected from: infectious bronchitis virus (IBV); guinea fowl coronavirus (GfCoV); and turkey coronavirus (TCoV; turkey enteritis virus and blue crown virus).

[0408] 42. The avian coronavirus spike protein or a fragment thereof of any one of technical claims 2 and 7 to 40, wherein at least a portion of the S2 subunit of an avian coronavirus having limited cell tropism or tissue tropism is derived from IBV (infectious bronchitis virus).

[0409] 43. The IBV spike protein or a fragment thereof of any one of claims 5 and 7 to 42, wherein at least a portion of the S2 subunit is derived from IBV selected from the following genotypes, serotypes, or strains: Arkansas (e.g., Arkansas 99), Brazil (e.g., BR-1, BR-2, 23 / 2013, IBV / Brasil / 351 / 1984), California (e.g., California 1734 / 04, California 99), Connecticut, Delaware (e.g., Delaware 98), Dutch (e.g., D207, D212, D274, D3128, D3896, D8880, D1466), Florida, Georgia (e.g., Georgia GA-07, GA-08, GA-12, GA-13), Gray, Holte, Iowa (e.g., Iowa 97 and Iowa 98). 69), Italy 02, JMK, LDT3, Maine (e.g., Maine 209), Massachusetts (e.g., H52, H120; excluding M41), Pennsylvania (e.g., Pennsylvania 1220 / 98, Pennsylvania Wolg / 98), PL84084, Qu (e.g., Qu-mv), QX (e.g., GB341 / 96), Q1, SE 17, variant 2 (e.g., IS / 1494 / 06, IBV / Ck / EG / CU / 4 / 2014, gammaCoV / Ck / Poland / G052 / 2016) and 4 / 91 (793B, CR88), or wherein at least a portion of the S2 subunit is not derived from IBV strain M41.

[0410] 44. The IBV spike protein or a fragment thereof of any one of claims 5 and 7 to 43, wherein at least a portion of the S2 subunit is derived from IBV selected from the following list of genotypes or serotypes or strains: Massachusetts (not M41), 4 / 91, QX, Q1, Italy O2, Arkansas, Conneticut, Georgia, LDT3, PL84084, variant 2, and Brazil.

[0411] 45. The IBV spike protein or a fragment thereof of any one of claims 5 and 7 to 43, wherein at least a portion of the S2 subunit is derived from IBV selected from the following list of genotypes or serotypes or strains: Massachusetts (not M41), 4 / 91, QX, Q1, Arkansas, variant 2, and Brazil.

[0412] 46. ​​The IBV spike protein or a fragment thereof of technical claim 45, wherein the Massachusetts strain is selected from the following list: H120, H52, Spain / 98 / 308, IBMA5-1, SD / 97 / 01 and Spain / 96 / 334.

[0413] 47. The IBV spike protein or a fragment thereof of technical claim 45, wherein the 4 / 91 strain is selected from the following list: Spain / 98 / 328, Spain / 92 / 35, IR-3654-VM, FR-CR88061-88, FR-85131-85, UK-1233-95, UK / 3 / 91, Spain / 00 / 336, UK / 7 / 91, pathogenic 4 / 91, attenuated 4 / 91, and IB4-91.

[0414] 48. The IBV spike protein or a fragment thereof of technical claim 45, wherein the QX strain is selected from the following list: FR-L1450T-05, FR-L1450L-05, NL-L1449T-04, NL-L1449K-04, IBV / Ck / SP / 170 / 09, IBV / Ck / SP / 79 / 08, IBV / Ck / SP / 248 / 09, HBN, IBVQX, LX4, BJQ, CK / CH / LGD / 03 and GB341 / 96.

[0415] 49. The IBV spike protein or a fragment thereof of technical claim 45, wherein the Q1 strain is selected from the following list: CK / CH / LDL / 98I, CK / CH / LSD / 08-10, J2, Q1, AR08ER22, AR08BA21 and Chile-295-10.

[0416] 50. The IBV spike protein or a fragment thereof of technical claim 45, wherein the Arkansas strain is selected from the following list: Ark99, ArkGA, ArkDPI, AL / 5364 / 00, ARKDPI11, AL / 0803 / 01, AL / 7149 / 00, ArkDPI101, AL / 1221 / 01, AL / 1793 / 01 and AL / 4614 / 98.

[0417] 51. The IBV spike protein or a fragment thereof of technical claim 45, wherein the variant 2 strain is selected from the following list: IS / 1494 / 06, IBV / Ck / EG / CU / 4 / 2014, gammaCoV / Ck / Poland / G052 / 2016, Eg / CLEVB-2 / IBV / 012, D1344 / 2 / 4 / 10_EG, TR8 and IB VAR2-06.

[0418] 52. The IBV spike protein or a fragment thereof of technical claim 45, wherein the Brazil strain is selected from the following list: BR-1, BR-2, 23 / 2013 and IBV / Brasil / 351 / 1984.

[0419] 53. The IBV spike protein or a fragment thereof of any one of claims 5 and 7 to 45, wherein at least a portion of the S2 subunit is derived from IBV selected from the following list of genotypes or serotypes: Massachusetts (not M41) and 4 / 91.

[0420] 54. The IBV spike protein or a fragment thereof of any one of technical solutions 5 and 7 to 45, wherein at least a portion of the S2 subunit is derived from IBV strains H120, H52 or CR88.

[0421] 55. The avian coronavirus spike protein or a fragment thereof of any one of claims 2 and 5 and 7 to 54, wherein at least a portion of the S2 subunit is an S2 subunit sequence of an avian coronavirus or IBV having limited cell tropism or tissue tropism, or at least 1, 5, 10, 15, 20, 25, 50, 100, 150, 200, 250, 300, 350, 400 or 500 adjacent amino acids of a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with it.

[0422] 56. The avian coronavirus or IBV spike protein or a fragment thereof of any one of claims 2 and 5 and 7 to 54, wherein at least a portion of the S2 subunit is an S2 subunit sequence of the avian coronavirus or IBV of any one of claims 36 to 50, or at least 1, 5, 10, 15, 20, 25, 50, 100, 150, 200, 250, 300, 350, 400 or 500 adjacent amino acids of a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with it.

[0423] 57. The IBV spike protein or a fragment thereof of any one of claims 5 and 7 to 54, wherein at least a portion of the S2 subunit of IBV having limited cell tropism or tissue tropism has an amino acid sequence as shown in SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, or at least 1, 5, 10, 15, 20, 25, 50, 100, 150, 200, 250, 300, 350, 400 or 500 adjacent amino acids of a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with it.

[0424] 58. The avian coronavirus or IBV spike protein or fragment thereof of any one of technical solutions 2 and 5 and 7 to 57, wherein the avian coronavirus or IBV with limited cell tropism or tissue tropism is limited to infection and / or replication in embryonic eggs and / or primary chicken kidney cells.

[0425] 59. The avian coronavirus or IBV spike protein or fragment thereof of any one of technical solutions 2 and 5 and 7 to 57, wherein the avian coronavirus or IBV with limited cell tropism or tissue tropism does not infect and / or replicate in EB66 and / or BHK cells.

[0426] 60. The avian coronavirus or IBV spike protein or fragment thereof of any one of technical solutions 2 and 5 and 7 to 57, wherein the avian coronavirus or IBV with limited cell tropism or tissue tropism does not infect and / or replicate in PBS-12 and / or HEK293T cells.

[0427] Excerpt

[0428] 61. The avian coronavirus or IBV spike protein or a fragment thereof of any one of technical solutions 1 to 60, wherein the fragment of said avian coronavirus or IBV spike protein has a length of at least 500, 750, 1000 or 1077 amino acids.

[0429] 62. The avian coronavirus or IBV spike protein or a fragment thereof of any one of technical solutions 1 to 61, wherein the fragment of the avian coronavirus or IBV spike protein has a length of at least 1,000 amino acids.

[0430] 63. The avian coronavirus or IBV spike protein of any one of technical solutions 1 to 62, wherein the aromatic amino acid at amino acid position 865 or the mutation to an aromatic amino acid at amino acid position 865 is genetically stable.

[0431] 64. A nucleotide sequence encoding a spike protein or a fragment thereof, which is any one of technical schemes 1 to 63.

[0432] 65. A plasmid comprising the nucleotide sequence of technical solution 64.

[0433] 66. A cell comprising the plasmid of technical solution 65.

[0434] 67. A viral particle comprising the spike protein or a fragment thereof of any one of technical solutions 1 to 63.

[0435] 68. An avian coronavirus comprising the spike protein or a fragment thereof of any one of technical solutions 1 to 63.

[0436] 69. An IBV (infectious bronchitis virus) comprising the spike protein of any one of claims 4 to 63.

[0437] 70. The avian coronavirus or IBV of technical solution 68 or 69, wherein the avian coronavirus or IBV is attenuated.

[0438] 71. The avian coronavirus or IBV of any one of technical solutions 68 to 70, wherein the avian coronavirus or IBV is genetically modified.

[0439] 72. The avian coronavirus or IBV of any one of technical solutions 68 to 71, wherein the avian coronavirus or IBV is recombinant.

[0440] 73. The avian coronavirus or IBV of any one of technical solutions 68 to 72, wherein the avian coronavirus or IBV is chimeric.

[0441] 74. An IBV of any one of technical solutions 69 to 73, wherein the IBV is derived from an IBV having a genotype or serotype or strain selected from the following list: Arkansas (e.g., Arkansas 99), Brazil (e.g., BR-1, BR-2, 23 / 2013, IBV / Brasil / 351 / 1984), California (e.g., California 1734 / 04, California 99), Connecticut, Delaware (e.g., Delaware 98), Dutch (e.g., D207, D212, D274, D3128, D3896, D8880, D1466), Florida, Georgia (e.g., Georgia GA-07, GA-08, GA-12, GA-13), Gray, Holte, Iowa (e.g., Iowa 97 and Iowa 69), Italy (e.g., Italy 02), JMK, LDT3, Maine (e.g., Maine 209), Massachusetts (M41, H52, H120, Beaudette), Pennsylvania (e.g., Pennsylvania 1220 / 98, Pennsylvania Wolg / 98), PL84084, Qu (e.g., Qu-mv), QX (e.g., GB341 / 96), Q1, SE 17, variant 2 (e.g., IS / 1494 / 06, IBV / Ck / EG / CU / 4 / 2014, gammaCoV / Ck / Poland / G052 / 2016) and 4 / 91 (793B, CR88).

[0442] 75. The IBV of any one of technical solutions 69 to 74, wherein the IBV is selected from the following list of genotypes or serotypes or strains: Massachusetts, 4 / 91, QX, Q1, Italy 02, Arkansas, Conneticut, Georgia, LDT3, PL84084, variant 2 and Brazil.

[0443] 76. The IBV of any one of technical solutions 69 to 75, wherein the IBV is selected from the following list of genotypes or serotypes: Massachusetts, 4 / 91, QX, Q1, Arkansas, variant 2 and Brazil.

[0444] 77. The IBV of technical solution 76, wherein the Massachusetts strain is selected from the following list: H120, H52, Spain / 98 / 308, IBMA5-1, SD / 97 / 01, Beaudette, Spain / 96 / 334 and M41-M21883.

[0445] 78. The IBV of technical solution 76, wherein the 4 / 91 strain is selected from the following list: Spain / 98 / 328, Spain / 92 / 35, IR-3654-VM, FR-CR88061-88, FR-85131-85, UK-1233-95, UK / 3 / 91, Spain / 00 / 336, UK / 7 / 91, pathogenic 4 / 91, attenuated 4 / 91 and IB4-91.

[0446] 79. The IBV of technical solution 76, wherein the QX strain is selected from the following list: FR-L1450T-05, FR-L1450L-05, NL-L1449T-04, NL-L1449K-04, IBV / Ck / SP / 170 / 09, IBV / Ck / SP / 79 / 08, IBV / Ck / SP / 248 / 09, HBN, IBVQX, LX4, BJQ, CK / CH / LGD / 03 and GB341 / 96.

[0447] 80. The IBV of technical solution 76, wherein the Q1 strain is selected from the following list: CK / CH / LDL / 98I, CK / CH / LSD / 08-10, J2, Q1, AR08ER22, AR08BA21 and Chile-295-10.

[0448] 81. The IBV of technical solution 76, wherein the Italy 02 strain is selected from the following list: Spain / 99 / 316, Italy-02, UK-L633-04, It-497-02, Spain / 05 / 866, Spain / 04 / 221, Spain / 00 / 337, Spain / 155 / 09 and Spain / 03 / 08.

[0449] 82. The IBV of technical solution 76, wherein the Arkansas strain is selected from the following list: Ark99, ArkGA, ArkDPI, AL / 5364 / 00, ARKDPI11, AL / 0803 / 01, AL / 7149 / 00, ArkDPI101, AL / 1221 / 01, AL / 1793 / 01 and AL / 4614 / 98.

[0450] 83. The IBV of technical solution 76, wherein the variant 2 strain is selected from the following list: IS / 1494 / 06, IBV / Ck / EG / CU / 4 / 2014, gammaCoV / Ck / Poland / G052 / 2016, Eg / CLEVB-2 / IBV / 012, D1344 / 2 / 4 / 10_EG, TR8 and IB VAR2-06.

[0451] 84. The IBV of technical claim 76, wherein the Brazil strain is selected from the following list: BR-1, BR-2, 23 / 2013 and IBV / Brasil / 351 / 1984.

[0452] 85. The IBV of any one of technical solutions 69 to 76, wherein the spike protein or a fragment thereof is derived from Massachusetts or IBV of genotype 4 / 91.

[0453] 86. The IBV of any one of technical solutions 69 to 76, wherein the IBV strain is H120, H52 or CR88.

[0454] 87. The IBV of any one of technical solutions 69 to 86, wherein the IBV comprises or includes the following IBV spike protein or fragments thereof: amino acid sequences as shown in SEQ ID NO:2, 3, 4, 5, 6, 7, 8, or sequences having at least 80%, 85%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98%, or 99.99% sequence identity with them.

[0455] 88. The IBV of any one of technical solutions 69 to 87, wherein the IBV has extended cell tropism or tissue tropism.

[0456] 89. The IBV of any one of technical solutions 69 to 88, wherein the IBV infects and / or replicates in at least one cell line or cell of any one of technical solutions 12 to 14.

[0457] 90. A cell comprising:

[0458] -Virus particles of technical solution 67, or

[0459] -Avian coronavirus or IBV from any of technical solutions 68 to 89.

[0460] 91. The cells of technical solution 90, wherein the cells are cell lines or cells selected from the following list: primary chicken embryo cells, chicken embryo fibroblast cell lines, duck embryo stem cell lines, human embryonic kidney cell lines, young hamster kidney cell lines, African green monkey kidney cell lines, rabbit kidney cell lines, canine kidney cell lines, chicken liver cell lines, bovine kidney cell lines, porcine kidney cell lines, and insect cell lines.

[0461] 92. Cells of technical claims 90 or 91, wherein the cells are cell lines selected from the following list: DF-1 (Douglas Foster), EB66 (duck embryo stem cell line), PBS-12, PBS-12SF (serum-free PBS-12), BHK21 (juvenile hamster kidney), HEK 293T (human embryo kidney), Vero (Verda Reno), MA104, RK13 (rabbit kidney), LMH (leghorn male liver cancer), MDCK (Martin Darby canine kidney), MDBK (Martin Darby bovine kidney), PK15 (pig kidney), PK2A (pig kidney), SF9, SF21, and SF+ (fall armyworm).

[0462] 93. The cells of any one of technical claims 90 to 92, wherein the cells are cell lines selected from the following list: DF-1, EB66, PBS-12, PBS-12SF, BHK, HEK 293T, Vero, MA104 and RK13.

[0463] 94. The cells of technical solution 91, wherein the primary chicken embryo cells are fibroblasts or cells derived from liver or lung tissue.

[0464] 95. An immunogenic composition comprising:

[0465] -The spike protein of any one of technical solutions 1 to 63, or

[0466] -Virus particles of technical solution 67, or

[0467] -Avian coronavirus or IBV from any of technical solutions 68 to 89.

[0468] 96. A vaccine comprising:

[0469] -The spike protein of any one of technical solutions 1 to 63, or

[0470] -Virus particles of technical solution 67, or

[0471] - Coronavirus or IBV from any of technical solutions 68 to 89.

[0472] 97. A modified live vaccine with extended cell tropism or tissue tropism, comprising:

[0473] -The spike protein of any one of technical solutions 1 to 63, or

[0474] -Virus particles of technical solution 67, or

[0475] - Coronavirus or IBV from any of technical solutions 68 to 89.

[0476] 98. An immunogenic composition or vaccine of any one of technical solutions 95 to 97, wherein the immunogenic composition or vaccine comprises a pharmaceutically acceptable carrier.

[0477] 99. The immunogenic composition or vaccine of technical solution 98, wherein the pharmaceutically acceptable carrier is phosphate-buffered saline.

[0478] 100. An immunogenic composition or vaccine of any one of technical claims 95 to 99, wherein the immunogenic composition or vaccine effectively treats and / or prevents clinical signs caused by IBV in subjects in need.

[0479] 101. An immunogenic composition or vaccine according to any one of technical solutions 95 to 100, wherein the immunogenic composition or vaccine comprises 1 to 10 log 10 EID 50 IBV.

[0480] 102. An immunogenic composition or vaccine according to any one of technical solutions 95 to 101, wherein the immunogenic composition or vaccine comprises 2 to 5 log 10 EID 50 IBV.

[0481] 103. An immunogenic composition or vaccine according to any one of technical solutions 95 to 102, wherein the immunogenic composition or vaccine comprises 2 to 4 log 10 EID 50 IBV.

[0482] 104. A method for altering the cell tropism or tissue tropism of avian coronavirus, comprising using the avian coronavirus spike protein or a fragment thereof from any one of technical solutions 1 to 63.

[0483] 105. A method for extending the cell tropism or tissue tropism of avian coronavirus, comprising using the avian coronavirus spike protein or a fragment thereof of any one of technical solutions 1 to 63.

[0484] 106. A method for producing or manufacturing avian coronaviruses with extended cell tropism or tissue tropism, comprising using the avian coronavirus spike protein or a fragment thereof from any one of technical solutions 1 to 63.

[0485] 107. A method for culturing avian coronavirus in cell culture or tissue culture, comprising using the avian coronavirus spike protein or a fragment thereof of any one of technical solutions 1 to 63.

[0486] 108. A method for modifying avian coronavirus, comprising modifying amino acid position 865 in the spike protein of the avian coronavirus.

[0487] 109. A method for mutating amino acid position 865 in the spike protein of avian coronavirus, comprising:

[0488] a) Provide the nucleotide or protein sequence of the avian coronavirus spike protein.

[0489] b) Identify position 865 in the spike protein by comparison with a reference sequence.

[0490] c) Mutate position 865 of the spike protein from step b) to an aromatic amino acid.

[0491] d) Obtain the mutated spike protein from step c).

[0492] 110. The method of any one of technical solutions 104 to 108, wherein the spike protein or a fragment thereof has an aromatic amino acid at amino acid position 865.

[0493] 111. The method of any one of technical solutions 108 to 110, wherein an aromatic amino acid at position 865 is introduced by mutation.

[0494] 112. The method of technical solution 111, wherein the mutation is an amino acid substitution, deletion or insertion.

[0495] 113. The method of any one of technical solutions 108 to 112, wherein the amino acid at amino acid position 865 is mutated to an aromatic amino acid; or the polar amino acid at amino acid position 865 is mutated to an aromatic amino acid; or the glutamine at amino acid position 865 is mutated to histidine.

[0496] 114. The method of any one of technical solutions 104 to 113, wherein the avian coronavirus is IBV of any one of technical solutions 63 to 83.

[0497] 115. The method of any one of technical solutions 104 to 114, wherein the coronavirus spike protein is the IBV (infectious bronchitis virus) spike protein of any one of technical solutions 3 to 57.

[0498] 116. The method of any one of technical solutions 104 to 115, wherein the aromatic amino acid at amino acid position 865 or the mutation at amino acid position 865 results in extended cell tropism or tissue tropism.

[0499] 117. The method of any one of technical solutions 104 to 116, wherein the avian coronavirus or IBV infects and / or replicates in at least one cell line or cell of any one of technical solutions 12 to 14.

[0500] 118. The method of any one of technical solutions 108 to 117, wherein the numbering of amino acid position 865 is completed according to any one of technical solutions 15 to 20.

[0501] reagent kit technical solution

[0502] 119. A kit comprising viral particles, avian coronavirus, IBV, immunogenic composition or vaccine of any one of technical solutions 67 to 89 and 95 to 103.

[0503] 120. The kit of technical solution 119, wherein the kit further comprises instructions for use in treating and / or preventing poultry diseases, or instructions for use in treating and / or preventing poultry diseases, or instructions for use in treating and / or preventing IB.

[0504] 121. The kit of technical solution 119 or 120, wherein the kit further includes a dispenser capable of administering the vaccine to the animal.

[0505] Treatment methods and technical solutions

[0506] 122. A method for immunizing a subject, comprising administering to such a subject an immunogenic composition or vaccine of any one of technical solutions 95 to 103.

[0507] 123. A method for treating or preventing clinical signs caused by IBV in a subject in need, the method comprising administering to the subject a therapeutically effective amount of an immunogenic composition or vaccine of any one of techniques 95 to 103.

[0508] 124. A method for reducing ciliary stagnation in a desired subject compared to a non-immunized control group of the same species, the method comprising administering to the subject a therapeutically effective amount of an immunogenic composition or vaccine of any one of technical solutions 95 to 103.

[0509] 125. An immunogenic composition or vaccine of any one of technical claims 95 to 103, wherein the method of immunizing a subject comprises administering a therapeutically effective amount of the immunogenic composition or vaccine to the subject.

[0510] 126. An immunogenic composition or vaccine of any one of technical claims 95 to 103, in a method for treating or preventing clinical signs caused by IBV in a subject of need, said method comprising administering a therapeutically effective amount of said immunogenic composition or vaccine to the subject.

[0511] 127. An immunogenic composition or vaccine of any one of claims 95 to 103, used in a method for reducing ciliary stagnation in a desired subject compared with a non-immunized control group of the same species, the method comprising administering a therapeutically effective amount of the immunogenic composition or vaccine to the subject.

[0512] 128. The method or use of any one of technical solutions 122 to 127, wherein the object is poultry.

[0513] 129. The method or use of any one of technical solutions 122 to 128, wherein the object is poultry.

[0514] 130. The method or use of any one of technical solutions 122 to 129, wherein the object is selected from chicken, turkey, quail or pheasant.

[0515] 131. The method or use of any one of technical solutions 122 to 130, wherein the object is a chicken.

[0516] 132. The method or use of any one of technical solutions 122 to 131, wherein the immunogenic composition or vaccine is administered once.

[0517] 133. The method or use of any one of technical solutions 122 to 131, wherein the immunogenic composition or vaccine is administered in two or more doses.

[0518] 134. The method or use of any one of technical solutions 122 to 133, wherein the immunogenic composition or vaccine is administered subcutaneously, intramuscularly, orally, intraovarianly, via spray, via drinking water, or via eye drops.

[0519] 135. The method or use of any one of technical solutions 122 to 134, wherein the immunogenic composition or vaccine is administered via eye drops.

[0520] 136. The method or use of any one of technical solutions 122 to 135, wherein the immunogenic composition or vaccine comprises 1 to 10 log 10 EID 50 / Dosage of IBV.

[0521] 137. The method or use of any one of technical solutions 122 to 136, wherein the immunogenic composition or vaccine comprises 2 to 5 log...10 EID 50 / Dosage of IBV.

[0522] 138. The method or use of any one of technical solutions 122 to 137, wherein the immunogenic composition or vaccine comprises 2 to 4 log 10 EID 50 / Dosage of IBV.

[0523] 139. The method or use of any one of technical solutions 122 to 138, wherein the immunogenic composition or vaccine is administered to a subject within the first week of life, within the first three days of life, within the first two days of life, or within the first day of life.

[0524] 140. The method or use of any one of technical solutions 122 to 139, wherein the immunogenic composition or vaccine is administered to a subject within the previous day of age.

[0525] 141. The method or use of any one of technical solutions 122 to 140, wherein the method results in an improvement in efficacy parameters selected from the following: prevention or reduction of ciliary arrest, prevention or reduction of rales, prevention or reduction of decreased egg production, prevention or reduction of kidney damage, prevention or reduction of watery diarrhea, prevention or reduction of weight loss, lower viral load, reduced viral shedding, or a combination thereof, compared with an untreated control group of the same species.

[0526] 142. The method or use of any one of technical solutions 122 to 141, wherein the treatment or prevention results in the prevention or reduction of ciliary arrest compared to an untreated control group of the same species.

[0527] 143. The method or use of any one of technical solutions 122 to 142, wherein the treatment or prevention results in the prevention or reduction of kidney damage compared to an untreated control group of the same species.

[0528] 144. The method or use of any one of technical solutions 122 to 143, wherein the treatment or prevention results in a reduction or decrease in egg production compared to an untreated control group of the same species.

[0529] 145. The viral particles, avian coronavirus, IBV, immunogenic composition or vaccine of any one of technical solutions 67 to 89 and 95 to 103, for therapeutic use.

[0530] 146. The viral particles, avian coronaviruses, or IBV of any one of technical solutions 67 to 89, which are used as immunogens or vaccines.

[0531] 147. The viral particles, avian coronavirus, IBV, immunogenic composition or vaccine of any one of technical solutions 67 to 89 and 95 to 103, used as a medicine.

[0532] 148. The use of any of the virus particles, avian coronaviruses, IBV, immunogenic compositions or vaccines of any of technical claims 67 to 89 and 95 to 103 for the manufacture of a pharmaceutical product.

[0533] 149. The use of any of the virus particles, avian coronaviruses, IBV, immunogenic compositions or vaccines of any of technical claims 67 to 89 and 95 to 103 for the treatment and / or prevention of IBV infection in subjects. sequence list <110> Boehringer Ingelheim Animal Health Ltd. <120> Modified S2 subunit of coronavirus spike protein <130> 01-3364 PCT <160> 31 <170> PatentIn version 3.5 <210> 1 <211> 1162 <212> PRT <213> Avian infectious bronchitis virus <400> 1 Met Leu Val Thr Pro Leu Leu Leu Val Thr Leu Leu Cys Ala Leu Cys 1 5 10 15 Ser Ala Ala Leu Tyr Asp Ser Ser Ser Tyr Val Tyr Tyr Tyr Gln Ser 20 25 30 Ala Phe Arg Pro Pro Asp Gly Trp His Leu His Gly Gly Ala Tyr Ala 35 40 45 Val Val Asn Ile Ser Ser Glu Ser Asn Asn Ala Gly Ser Ser Ser Gly 50 55 60 Cys Thr Val Gly Ile Ile His Gly Gly Arg Val Val Asn Ala Ser Ser 65 70 75 80 Ile Ala Met Thr Ala Pro Ser Ser Gly Met Ala Trp Ser Ser Ser Gln 85 90 95 Phe Cys Thr Ala Tyr Cys Asn Phe Ser Asp Thr Thr Val Phe Val Thr 100 105 110 His Cys Tyr Lys His Gly Gly Cys Pro Ile Thr Gly Met Leu Gln Gln 115 120 125 His Ser Ile Arg Val Ser Ala Met Lys Asn Gly Gln Leu Phe Tyr Asn 130 135 140 Leu Thr Val Ser Val Ala Lys Tyr Pro Thr Phe Lys Ser Phe Gln Cys 145 150 155 160 Val Asn Asn Leu Thr Ser Val Tyr Leu Asn Gly Asp Leu Val Tyr Thr 165 170 175 Ser Asn Glu Thr Thr Asp Val Thr Ser Ala Gly Val Tyr Phe Lys Ala 180 185 190 Gly Gly Pro Ile Thr Tyr Lys Val Met Arg Glu Val Arg Ala Leu Ala 195 200 205 Tyr Phe Val Asn Gly Thr Ala Gln Asp Val Ile Leu Cys Asp Gly Ser 210 215 220 Pro Arg Gly Leu Leu Ala Cys Gln Tyr Asn Thr Gly Asn Phe Ser Asp 225 230 235 240 Gly Phe Tyr Pro Phe Thr Asn Ser Ser Leu Val Lys Gln Lys Phe Ile 245 250 255 Val Tyr Arg Glu Asn Ser Val Asn Thr Thr Phe Thr Leu His Asn Phe 260 265 270 Thr Phe His Asn Glu Thr Gly Ala Asn Pro Asn Pro Ser Gly Val Gln 275 280 285 Asn Ile Gln Thr Tyr Gln Thr Gln Thr Ala Gln Ser Gly Tyr Tyr Asn 290 295 300 Phe Asn Phe Ser Phe Leu Ser Ser Phe Val Tyr Lys Glu Ser Asn Phe 305 310 315 320 Met Tyr Gly Ser Tyr His Pro Ser Cys Asn Phe Arg Leu Glu Thr Ile 325 330 335 Asn Asn Gly Leu Trp Phe Asn Ser Leu Ser Val Ser Ile Ala Tyr Gly 340 345 350 Pro Leu Gln Gly Gly Cys Lys Gln Ser Val Phe Ser Gly Arg Ala Thr 355 360 365 Cys Cys Tyr Ala Tyr Ser Tyr Gly Gly Pro Leu Leu Cys Lys Gly Val 370 375 380 Tyr Ser Gly Glu Leu Asp His Asn Phe Glu Cys Gly Leu Leu Val Tyr 385 390 395 400 Val Thr Lys Ser Gly Gly Ser Arg Ile Gln Thr Ala Thr Glu Pro Pro 405 410 415 Val Ile Thr Gln His Asn Tyr Asn Asn Ile Thr Leu Asn Thr Cys Val 420 425 430 Asp Tyr Asn Ile Tyr Gly Arg Thr Gly Gln Gly Phe Ile Thr Asn Val 435 440 445 Thr Asp Ser Ala Val Ser Tyr Asn Tyr Leu Ala Asp Ala Gly Leu Ala 450 455 460 Ile Leu Asp Thr Ser Gly Ser Ile Asp Ile Phe Val Val Gln Ser Glu 465 470 475 480 Tyr Gly Leu Asn Tyr Tyr Lys Val Asn Pro Cys Glu Asp Val Asn Gln 485 490 495 Gln Phe Val Val Ser Gly Gly Lys Leu Val Gly Ile Leu Thr Ser Arg 500 505 510 Asn Glu Thr Gly Ser Gln Leu Leu Glu Asn Gln Phe Tyr Ile Lys Ile 515 520 525 Thr Asn Gly Thr Arg Arg Phe Arg Arg Ser Ile Thr Glu Ser Val Glu 530 535 540 Asn Cys Pro Tyr Val Ser Tyr Gly Lys Phe Cys Ile Lys Pro Asp Gly 545 550 555 560 Ser Ile Ala Thr Ile Val Pro Lys Gln Leu Glu Gln Phe Val Ala Pro 565 570 575 Leu Leu Asn Val Thr Glu Asn Val Leu Ile Pro Asn Ser Phe Asn Leu 580 585 590 Thr Val Thr Asp Glu Tyr Ile Gln Thr Arg Met Asp Lys Val Gln Ile 595 600 605 Asn Cys Leu Gln Tyr Ile Cys Gly Asn Ser Leu Glu Cys Arg Asn Leu 610 615 620 Phe Gln Gln Tyr Gly Pro Val Cys Asp Asn Ile Leu Ser Val Val Asn 625 630 635 640 Ser Val Gly Gln Lys Glu Asp Met Glu Leu Leu Asn Phe Tyr Ser Ser 645 650 655 Thr Lys Pro Ala Gly Phe Asn Thr Pro Val Leu Ser Asn Val Ser Thr 660 665 670 Gly Glu Phe Asn Ile Thr Leu Phe Leu Thr Thr Pro Ser Ser Pro Arg 675 680 685 Arg Arg Ser Phe Ile Glu Asp Leu Leu Phe Thr Ser Val Glu Ser Val 690 695 700 Gly Leu Pro Thr Asp Asp Ala Tyr Lys Asn Cys Thr Ala Gly Pro Leu 705 710 715 720 Gly Phe Leu Lys Asp Leu Ala Cys Ala Arg Glu Tyr Asn Gly Leu Leu 725 730 735 Val Leu Pro Pro Ile Ile Thr Ala Glu Met Gln Thr Leu Tyr Thr Ser 740 745 750 Ser Leu Val Ala Ser Met Ala Phe Gly Gly Ile Thr Ala Ala Gly Ala 755 760 765 Ile Pro Phe Ala Thr Gln Leu Gln Ala Arg Ile Asn His Leu Gly Ile 770 775 780 Thr Gln Ser Leu Leu Leu Lys Asn Gln Glu Lys Ile Ala Ala Ser Phe 785 790 795 800 Asn Lys Ala Ile Gly His Met Gln Glu Gly Phe Arg Ser Thr Ser Leu 805 810 815 Ala Leu Gln Gln Ile Gln Asp Val Val Asn Lys Gln Ser Ala Ile Leu 820 825 830 Thr Glu Thr Met Ala Ser Leu Asn Lys Asn Phe Gly Ala Ile Ser Ser 835 840 845 Val Ile Gln Glu Ile Tyr Gln Gln Leu Asp Ala Ile Gln Ala Asn Ala 850 855 860 Gln Val Asp Arg Leu Ile Thr Gly Arg Leu Ser Ser Leu Ser Val Leu 865 870 875 880 Ala Ser Ala Lys Gln Ala Glu Tyr Ile Arg Val Ser Gln Gln Arg Glu 885 890 895 Leu Ala Thr Gln Lys Ile Asn Glu Cys Val Lys Ser Gln Ser Ile Arg 900 905 910 Tyr Ser Phe Cys Gly Asn Gly Arg His Val Leu Thr Ile Pro Gln Asn 915 920 925 Ala Pro Asn Gly Ile Val Phe Ile His Phe Ser Tyr Thr Pro Asp Ser 930 935 940 Phe Val Asn Val Thr Ala Ile Val Gly Phe Cys Val Lys Pro Ala Asn 945 950 955 960 Ala Ser Gln Tyr Ala Ile Val Pro Ala Asn Gly Arg Gly Ile Phe Ile 965 970 975 Gln Val Asn Gly Ser Tyr Tyr Ile Thr Ala Arg Asp Met Tyr Met Pro 980 985 990 Arg Ala Ile Thr Ala Gly Asp Ile Val Thr Leu Thr Ser Cys Gln Ala 995 1000 1005 Asn Tyr Val Ser Val Asn Lys Thr Val Ile Thr Thr Phe Val Asp 1010 1015 1020 Asn Asp Asp Phe Asp Phe Asn Asp Glu Leu Ser Lys Trp Trp Asn 1025 1030 1035 Asp Thr Lys His Glu Leu Pro Asp Phe Asp Lys Phe Asn Tyr Thr 1040 1045 1050 Val Pro Ile Leu Asp Ile Asp Ser Glu Ile Asp Arg Ile Gln Gly 1055 1060 1065 Val Ile Gln Gly Leu Asn Asp Ser Leu Ile Asp Leu Glu Lys Leu 1070 1075 1080 Ser Ile Leu Lys Thr Tyr Ile Lys Trp Pro Trp Tyr Val Trp Leu 1085 1090 1095 Ala Ile Ala Phe Ala Thr Ile Ile Phe Ile Leu Ile Leu Gly Trp 1100 1105 1110 Val Phe Phe Met Thr Gly Cys Cys Gly Cys Cys Cys Gly Cys Phe 1115 1120 1125 Gly Ile Met Pro Leu Met Ser Lys Cys Gly Lys Lys Ser Ser Tyr 1130 1135 1140 Tyr Thr Thr Phe Asp Asn Asp Val Val Thr Glu Gln Tyr Arg Pro 1145 1150 1155 Lys Lys Ser Val 1160 <210> 2 <211> 1162 <212> PRT <213> Avian infectious bronchitis virus <400> 2 Met Leu Val Thr Pro Leu Leu Leu Val Thr Leu Leu Cys Ala Leu Cys 1 5 10 15 Ser Ala Ala Leu Tyr Asp Ser Ser Ser Tyr Val Tyr Tyr Tyr Gln Ser 20 25 30 Ala Phe Arg Pro Pro Asp Gly Trp His Leu His Gly Gly Ala Tyr Ala 35 40 45 Val Val Asn Ile Ser Ser Glu Ser Asn Asn Ala Gly Ser Ser Ser Gly 50 55 60 Cys Thr Val Gly Ile Ile His Gly Gly Arg Val Val Asn Ala Ser Ser 65 70 75 80 Ile Ala Met Thr Ala Pro Ser Ser Gly Met Ala Trp Ser Ser Ser Gln 85 90 95 Phe Cys Thr Ala Tyr Cys Asn Phe Ser Asp Thr Thr Val Phe Val Thr 100 105 110 His Cys Tyr Lys His Gly Gly Cys Pro Ile Thr Gly Met Leu Gln Gln 115 120 125 His Ser Ile Arg Val Ser Ala Met Lys Asn Gly Gln Leu Phe Tyr Asn 130 135 140 Leu Thr Val Ser Val Ala Lys Tyr Pro Thr Phe Lys Ser Phe Gln Cys 145 150 155 160 Val Asn Asn Leu Thr Ser Val Tyr Leu Asn Gly Asp Leu Val Tyr Thr 165 170 175 Ser Asn Glu Thr Thr Asp Val Thr Ser Ala Gly Val Tyr Phe Lys Ala 180 185 190 Gly Gly Pro Ile Thr Tyr Lys Val Met Arg Glu Val Arg Ala Leu Ala 195 200 205 Tyr Phe Val Asn Gly Thr Ala Gln Asp Val Ile Leu Cys Asp Gly Ser 210 215 220 Pro Arg Gly Leu Leu Ala Cys Gln Tyr Asn Thr Gly Asn Phe Ser Asp 225 230 235 240 Gly Phe Tyr Pro Phe Thr Asn Ser Ser Leu Val Lys Gln Lys Phe Ile 245 250 255 Val Tyr Arg Glu Asn Ser Val Asn Thr Thr Phe Thr Leu His Asn Phe 260 265 270 Thr Phe His Asn Glu Thr Gly Ala Asn Pro Asn Pro Ser Gly Val Gln 275 280 285 Asn Ile Gln Thr Tyr Gln Thr Gln Thr Ala Gln Ser Gly Tyr Tyr Asn 290 295 300 Phe Asn Phe Ser Phe Leu Ser Ser Phe Val Tyr Lys Glu Ser Asn Phe 305 310 315 320 Met Tyr Gly Ser Tyr His Pro Ser Cys Asn Phe Arg Leu Glu Thr Ile 325 330 335 Asn Asn Gly Leu Trp Phe Asn Ser Leu Ser Val Ser Ile Ala Tyr Gly 340 345 350 Pro Leu Gln Gly Gly Cys Lys Gln Ser Val Phe Ser Gly Arg Ala Thr 355 360 365 Cys Cys Tyr Ala Tyr Ser Tyr Gly Gly Pro Leu Leu Cys Lys Gly Val 370 375 380 Tyr Ser Gly Glu Leu Asp His Asn Phe Glu Cys Gly Leu Leu Val Tyr 385 390 395 400 Val Thr Lys Ser Gly Gly Ser Arg Ile Gln Thr Ala Thr Glu Pro Pro 405 410 415 Val Ile Thr Gln His Asn Tyr Asn Asn Ile Thr Leu Asn Thr Cys Val 420 425 430 Asp Tyr Asn Ile Tyr Gly Arg Thr Gly Gln Gly Phe Ile Thr Asn Val 435 440 445 Thr Asp Ser Ala Val Ser Tyr Asn Tyr Leu Ala Asp Ala Gly Leu Ala 450 455 460 Ile Leu Asp Thr Ser Gly Ser Ile Asp Ile Phe Val Val Gln Ser Glu 465 470 475 480 Tyr Gly Leu Asn Tyr Tyr Lys Val Asn Pro Cys Glu Asp Val Asn Gln 485 490 495 Gln Phe Val Val Ser Gly Gly Lys Leu Val Gly Ile Leu Thr Ser Arg 500 505 510 Asn Glu Thr Gly Ser Gln Leu Leu Glu Asn Gln Phe Tyr Ile Lys Ile 515 520 525 Thr Asn Gly Thr Arg Arg Phe Arg Arg Ser Ile Thr Glu Ser Val Glu 530 535 540 Asn Cys Pro Tyr Val Ser Tyr Gly Lys Phe Cys Ile Lys Pro Asp Gly 545 550 555 560 Ser Ile Ala Thr Ile Val Pro Lys Gln Leu Glu Gln Phe Val Ala Pro 565 570 575 Leu Leu Asn Val Thr Glu Asn Val Leu Ile Pro Asn Ser Phe Asn Leu 580 585 590 Thr Val Thr Asp Glu Tyr Ile Gln Thr Arg Met Asp Lys Val Gln Ile 595 600 605 Asn Cys Leu Gln Tyr Ile Cys Gly Asn Ser Leu Glu Cys Arg Asn Leu 610 615 620 Phe Gln Gln Tyr Gly Pro Val Cys Asp Asn Ile Leu Ser Val Val Asn 625 630 635 640 Ser Val Gly Gln Lys Glu Asp Met Glu Leu Leu Asn Phe Tyr Ser Ser 645 650 655 Thr Lys Pro Ala Gly Phe Asn Thr Pro Val Leu Ser Asn Val Ser Thr 660 665 670 Gly Glu Phe Asn Ile Thr Leu Phe Leu Thr Thr Pro Ser Ser Pro Arg 675 680 685 Arg Arg Ser Phe Ile Glu Asp Leu Leu Phe Thr Ser Val Glu Ser Val 690 695 700 Gly Leu Pro Thr Asp Asp Ala Tyr Lys Asn Cys Thr Ala Gly Pro Leu 705 710 715 720 Gly Phe Leu Lys Asp Leu Ala Cys Ala Arg Glu Tyr Asn Gly Leu Leu 725 730 735 Val Leu Pro Pro Ile Ile Thr Ala Glu Met Gln Thr Leu Tyr Thr Ser 740 745 750 Ser Leu Val Ala Ser Met Ala Phe Gly Gly Ile Thr Ala Ala Gly Ala 755 760 765 Ile Pro Phe Ala Thr Gln Leu Gln Ala Arg Ile Asn His Leu Gly Ile 770 775 780 Thr Gln Ser Leu Leu Leu Lys Asn Gln Glu Lys Ile Ala Ala Ser Phe 785 790 795 800 Asn Lys Ala Ile Gly His Met Gln Glu Gly Phe Arg Ser Thr Ser Leu 805 810 815 Ala Leu Gln Gln Ile Gln Asp Val Val Asn Lys Gln Ser Ala Ile Leu 820 825 830 Thr Glu Thr Met Ala Ser Leu Asn Lys Asn Phe Gly Ala Ile Ser Ser 835 840 845 Val Ile Gln Glu Ile Tyr Gln Gln Leu Asp Ala Ile Gln Ala Asn Ala 850 855 860 His Val Asp Arg Leu Ile Thr Gly Arg Leu Ser Ser Leu Ser Val Leu 865 870 875 880 Ala Ser Ala Lys Gln Ala Glu Tyr Ile Arg Val Ser Gln Gln Arg Glu 885 890 895 Leu Ala Thr Gln Lys Ile Asn Glu Cys Val Lys Ser Gln Ser Ile Arg 900 905 910 Tyr Ser Phe Cys Gly Asn Gly Arg His Val Leu Thr Ile Pro Gln Asn 915 920 925 Ala Pro Asn Gly Ile Val Phe Ile His Phe Ser Tyr Thr Pro Asp Ser 930 935 940 Phe Val Asn Val Thr Ala Ile Val Gly Phe Cys Val Lys Pro Ala Asn 945 950 955 960 Ala Ser Gln Tyr Ala Ile Val Pro Ala Asn Gly Arg Gly Ile Phe Ile 965 970 975 Gln Val Asn Gly Ser Tyr Tyr Ile Thr Ala Arg Asp Met Tyr Met Pro 980 985 990 Arg Ala Ile Thr Ala Gly Asp Ile Val Thr Leu Thr Ser Cys Gln Ala 995 1000 1005 Asn Tyr Val Ser Val Asn Lys Thr Val Ile Thr Thr Phe Val Asp 1010 1015 1020 Asn Asp Asp Phe Asp Phe Asn Asp Glu Leu Ser Lys Trp Trp Asn 1025 1030 1035 Asp Thr Lys His Glu Leu Pro Asp Phe Asp Lys Phe Asn Tyr Thr 1040 1045 1050 Val Pro Ile Leu Asp Ile Asp Ser Glu Ile Asp Arg Ile Gln Gly 1055 1060 1065 Val Ile Gln Gly Leu Asn Asp Ser Leu Ile Asp Leu Glu Lys Leu 1070 1075 1080 Ser Ile Leu Lys Thr Tyr Ile Lys Trp Pro Trp Tyr Val Trp Leu 1085 1090 1095 Ala Ile Ala Phe Ala Thr Ile Ile Phe Ile Leu Ile Leu Gly Trp 1100 1105 1110 Val Phe Phe Met Thr Gly Cys Cys Gly Cys Cys Cys Gly Cys Phe 1115 1120 1125 Gly Ile Met Pro Leu Met Ser Lys Cys Gly Lys Lys Ser Ser Tyr 1130 1135 1140 Tyr Thr Thr Phe Asp Asn Asp Val Val Thr Glu Gln Tyr Arg Pro 1145 1150 1155 Lys Lys Ser Val 1160 <210> 3 <211> 1155 <212> PRT <213> Avian infectious bronchitis virus <400> 3 Met Leu Asp Lys Pro Leu Leu Leu Val Thr Leu Trp Tyr Ala Leu Cys 1 5 10 15 Ser Ala Leu Leu Tyr Asp Asn Asn Thr Tyr Val Tyr Tyr Tyr Gln Ser 20 25 30 Ala Phe Arg Pro Gly Pro Gly Trp His Leu Tyr Gly Gly Ala Tyr Ala 35 40 45 Val Asp Arg Val Phe Asn Glu Thr Asn Asn Ala Gly Ser Ala Ser Asp 50 55 60 Cys Thr Ala Gly Thr Phe Tyr Glu Ser His Asn Ile Ser Ala Ser Ser 65 70 75 80 Val Ala Met Thr Val Pro His Asn Gly Met Ser Trp Ser Ala Ser Gln 85 90 95 Phe Cys Thr Ala His Cys Asn Phe Ser Asp Phe Thr Val Phe Val Thr 100 105 110 His Cys Phe Lys Asn Gln Leu Gly Ser Cys Pro Leu Thr Gly Met Ile 115 120 125 Pro Gln Asn His Ile Arg Ile Ser Ala Met Arg Asp Gly Val Leu Phe 130 135 140 Tyr Asn Leu Thr Val Ser Val Ser Lys Tyr Pro Arg Phe Lys Ser Leu 145 150 155 160 Gln Cys Val Ser Asn Ser Thr Ser Val Tyr Val Asn Gly Asp Leu Val 165 170 175 Phe Thr Ser Asn Glu Thr Ser Tyr Val Thr Gly Ala Gly Val Tyr Phe 180 185 190 Lys Ser Gly Gly Pro Val Thr Tyr Lys Val Met Lys Glu Val Lys Ala 195 200 205 Leu Ala Tyr Phe Ile Asn Gly Thr Ala Gln Glu Val Ile Leu Cys Asp 210 215 220 Asn Ser Pro Arg Gly Leu Leu Ala Cys Gln Tyr Asn Thr Gly Asn Phe 225 230 235 240 Ser Asp Gly Phe Tyr Pro Phe Thr Asn His Ser Leu Val Lys Asp Arg 245 250 255 Phe Ile Val Tyr Arg Glu Ser Ser Thr Asn Thr Thr Leu Lys Leu Thr 260 265 270 Asn Phe Ser Phe Thr Asn Val Ser Asn Ala Ser Pro Asn Ser Gly Gly 275 280 285 Val Asp Thr Phe Gln Leu Tyr Gln Thr Ser Thr Ala Gln Asp Gly Tyr 290 295 300 Tyr Asn Phe Asn Leu Ser Phe Leu Ser Ser Phe Val Tyr Lys Pro Ser 305 310 315 320 Asp Phe Met Tyr Gly Ser Tyr His Pro His Cys Lys Phe Arg Pro Glu 325 330 335 Asn Ile Asn Asn Gly Leu Trp Phe Asn Ser Leu Ser Val Ser Leu Thr 340 345 350 Tyr Gly Pro Ile Gln Gly Gly Cys Lys Gln Ser Val Phe Ser Asn Arg 355 360 365 Ala Thr Cys Cys Tyr Ala Tyr Ser Tyr Gln Gly Pro Ser Arg Cys Lys 370 375 380 Gly Val Tyr Arg Gly Glu Leu Thr Gln Tyr Phe Glu Cys Gly Leu Leu 385 390 395 400 Val Tyr Val Thr Lys Ser Asp Gly Ser Arg Ile Gln Thr Arg Ser Glu 405 410 415 Pro Leu Val Leu Thr Gln Tyr Asn Tyr Asn Asn Ile Thr Leu Asn Lys 420 425 430 Cys Val Glu Tyr Asn Ile Tyr Gly Arg Val Gly Gln Gly Phe Ile Thr 435 440 445 Asn Val Thr Glu Ala Thr Ala Asn Tyr Ser Tyr Leu Ala Asp Gly Gly 450 455 460 Leu Ala Ile Leu Asp Thr Ser Gly Ala Ile Asp Ile Phe Val Val Gln 465 470 475 480 Gly Ala Tyr Gly Leu Asn Tyr Tyr Lys Val Asn Pro Cys Glu Asp Val 485 490 495 Asn Gln Gln Phe Val Val Ser Gly Gly Asn Leu Val Gly Ile Leu Thr 500 505 510 Ser His Asn Glu Thr Gly Ser Glu Ser Ile Glu Asn Gln Phe Tyr Ile 515 520 525 Lys Leu Thr Asn Gly Thr Arg Arg Ser Arg Arg Ser Val Thr Gly Asn 530 535 540 Val Thr Asn Cys Pro Tyr Val Ser Tyr Gly Lys Phe Cys Ile Lys Pro 545 550 555 560 Asp Gly Ser Leu Ser Ile Ile Val Pro Gln Glu Leu Glu Gln Phe Val 565 570 575 Ala Pro Leu Phe Asn Val Thr Glu His Val Leu Ile Pro Asp Ser Phe 580 585 590 Asn Leu Thr Val Thr Asp Glu Tyr Ile Gln Thr Arg Met Asp Lys Val 595 600 605 Gln Ile Ile Cys Leu Gln Tyr Val Cys Gly Asn Ser Ile Glu Cys Arg 610 615 620 Lys Leu Phe Gln Gln Tyr Gly Pro Val Cys Asp Asn Ile Leu Ser Val 625 630 635 640 Val Asn Gly Val Gly Gln Arg Glu Asp Met Glu Leu Leu Ser Phe Tyr 645 650 655 Ser Ser Thr Lys Pro Ser Gly Tyr Asn Thr Pro Ile Phe Asn Asn Val 660 665 670 Ser Thr Gly Asp Phe Asn Ile Ser Leu Leu Leu Thr Pro Pro Asn Ser 675 680 685 Pro Thr Gly Arg Ser Phe Ile Glu Asp Leu Leu Phe Thr Ser Val Glu 690 695 700 Ser Val Gly Leu Pro Thr Asp Glu Glu Tyr Lys Lys Cys Thr Ala Gly 705 710 715 720 Pro Leu Gly Phe Val Lys Asp Leu Val Cys Ala Arg Glu Tyr Asn Gly 725 730 735 Leu Leu Val Leu Pro Pro Ile Ile Thr Ala Glu Met Gln Thr Met Tyr 740 745 750 Thr Ser Ser Leu Val Ala Ser Met Ala Leu Gly Gly Ile Thr Ala Ala 755 760 765 Gly Ala Ile Pro Phe Ala Thr Gln Leu Gln Ala Arg Ile Asn His Leu 770 775 780 Gly Ile Thr Asn Ser Leu Leu Leu Lys Asn Gln Glu Lys Ile Ala Ala 785 790 795 800 Ser Phe Asn Lys Ala Ile Gly His Met Gln Glu Gly Phe Lys Ser Thr 805 810 815 Ser Leu Ala Leu Gln Gln Ile Gln Asp Val Val Asn Lys Gln Ser Ser 820 825 830 Ile Leu Thr Glu Thr Met Gln Ser Leu Asn Lys Asn Phe Gly Ala Ile 835 840 845 Ser Ser Val Ile Gln Asp Ile Tyr Gln Gln Leu Asp Ala Ile Gln Ala 850 855 860 Asp Ala His Val Asp Arg Leu Ile Thr Gly Arg Leu Ser Ser Leu Ser 865 870 875 880 Val Leu Ala Ser Ala Lys Gln Ala Glu Tyr His Arg Val Ser Gln Gln 885 890 895 Arg Glu Leu Ala Thr Gln Lys Ile Asn Glu Cys Val Lys Ser Gln Ser 900 905 910 Asn Arg Tyr Ser Phe Cys Gly Asn Gly Arg His Val Leu Thr Ile Pro 915 920 925 Gln Asn Ala Pro Asn Gly Ile Val Phe Ile His Phe Thr Tyr Thr Pro 930 935 940 Glu Ser Phe Val Asn Val Thr Ala Ile Val Gly Phe Cys Val Asn Pro 945 950 955 960 Ala Asn Ala Ser His Tyr Ala Ile Val Pro Val Asn Gly Arg Gly Val 965 970 975 Phe Ile Glu Val Asn Gly Ser Tyr Tyr Ile Thr Ala Arg Asp Met Tyr 980 985 990 Met Pro Arg Asp Ile Thr Ala Gly Asp Ile Val Thr Leu Thr Ser Cys 995 1000 1005 Gln Ala Asn Tyr Val Asn Val Asn Lys Thr Val Ile Asn Thr Phe 1010 1015 1020 Val Glu Asp Asp Asp Phe Asp Phe Tyr Asp Glu Leu Ser Lys Trp 1025 1030 1035 Trp Asn Asp Thr Lys His Glu Leu Pro Asp Phe Asp Glu Phe Asn 1040 1045 1050 Tyr Thr Val Pro Val Leu Asn Ile Ser Asn Glu Ile Asp Arg Ile 1055 1060 1065 Gln Gln Val Ile Gln Gly Leu Asn Asp Ser Leu Ile Asp Leu Glu 1070 1075 1080 Thr Leu Ser Ile Leu Lys Thr Tyr Ile Lys Trp Pro Trp Tyr Val 1085 1090 1095 Trp Leu Ala Ile Ala Phe Leu Thr Ile Ile Phe Ile Leu Val Leu 1100 1105 1110 Cys Trp Ile Phe Phe Met Thr Gly Cys Cys Gly Cys Cys Cys Gly 1115 1120 1125 Cys Phe Gly Ile Ile Pro Leu Met Ser Lys Cys Gly Lys Lys Ser 1130 1135 1140 Ser Tyr Tyr Thr Thr Phe Asp Asn Asp Val Val Thr 1145 1150 1155 <210> 4 <211> 1156 <212> PRT <213> Avian infectious bronchitis virus <400> 4 Met Leu Val Lys Ser Leu Phe Leu Val Thr Ile Leu Cys Ala Leu Cys 1 5 10 15 Ser Ala Asn Leu Phe Asp Ser Asp Asn Asn Tyr Val Tyr Tyr Tyr Gln 20 25 30 Ser Ala Phe Arg Pro Pro Asn Gly Trp His Leu Gln Gly Gly Ala Tyr 35 40 45 Ala Val Val Ser Ser Thr Asn Tyr Thr Asn Asn Ala Gly Ser Ala His 50 55 60 Gly Cys Thr Val Gly Val Ile Lys Asp Val Tyr Asn Gln Ser Val Ala 65 70 75 80 Ser Ile Ala Met Thr Ala Pro Leu Gln Gly Met Ala Trp Ser Lys Ser 85 90 95 Gln Phe Cys Ser Ala His Cys Asn Phe Ser Glu Ile Thr Val Phe Val 100 105 110 Thr His Cys Tyr Ser Ser Gly Ser Gly Ser Cys Pro Ile Thr Gly Met 115 120 125 Ile Pro Arg Asp His Ile Arg Ile Ser Ala Met Lys Asn Gly Ser Leu 130 135 140 Phe Tyr Asn Leu Thr Val Ser Val Ser Lys Tyr Pro Asn Phe Lys Ser 145 150 155 160 Phe Gln Cys Val Asn Asn Phe Thr Ser Val Tyr Leu Asn Gly Asp Leu 165 170 175 Val Phe Thr Ser Asn Lys Thr Thr Asp Val Thr Ser Ala Gly Val Tyr 180 185 190 Phe Lys Ala Gly Gly Pro Val Asn Tyr Ser Ile Met Lys Glu Phe Lys 195 200 205 Val Leu Ala Tyr Phe Val Asn Gly Thr Ala Gln Asp Val Val Leu Cys 210 215 220 Asp Asn Ser Pro Lys Gly Leu Leu Ala Cys Gln Tyr Asn Thr Gly Asn 225 230 235 240 Phe Ser Asp Gly Phe Tyr Pro Phe Thr Asn Ser Thr Leu Val Arg Glu 245 250 255 Lys Phe Ile Val Tyr Arg Glu Ser Ser Val Asn Thr Thr Leu Ala Leu 260 265 270 Thr Asn Phe Thr Phe Ser Asn Val Ser Asn Ala Gln Pro Asn Ser Gly 275 280 285 Gly Val Asn Thr Phe His Leu Tyr Gln Thr Gln Thr Ala Gln Ser Gly 290 295 300 Tyr Tyr Asn Phe Asn Leu Ser Phe Leu Ser Gln Phe Val Tyr Lys Ala 305 310 315 320 Ser Asp Phe Met Tyr Gly Ser Tyr His Pro Ser Cys Ser Phe Arg Pro 325 330 335 Glu Thr Ile Asn Ser Gly Leu Trp Phe Asn Ser Leu Ser Val Ser Leu 340 345 350 Thr Tyr Gly Pro Leu Gln Gly Gly Cys Lys Gln Ser Val Phe Ser Gly 355 360 365 Lys Ala Thr Cys Cys Tyr Ala Tyr Ser Tyr Arg Gly Pro Met Ala Cys 370 375 380 Lys Gly Val Tyr Ser Gly Glu Leu Ser Thr Asn Phe Glu Cys Gly Leu 385 390 395 400 Leu Val Tyr Val Thr Lys Ser Asp Gly Ser Arg Ile Gln Thr Arg Thr 405 410 415 Glu Pro Leu Val Leu Thr Gln Tyr Asn Tyr Asn Asn Ile Thr Leu Asp 420 425 430 Lys Cys Val Ala Tyr Asn Ile Tyr Gly Arg Val Gly Gln Gly Phe Ile 435 440 445 Thr Asn Val Thr Asp Ser Ala Ala Asn Phe Ser Tyr Leu Ala Asp Gly 450 455 460 Gly Leu Ala Ile Leu Asp Thr Ser Gly Ala Ile Asp Val Phe Val Val 465 470 475 480 Gln Gly Ile Tyr Gly Leu Asn Tyr Tyr Lys Val Asn Pro Cys Glu Asp 485 490 495 Val Asn Gln Gln Phe Val Val Ser Gly Gly Asn Ile Val Gly Ile Leu 500 505 510 Thr Ser Arg Asn Glu Thr Gly Ser Glu Gln Val Glu Asn Gln Phe Tyr 515 520 525 Val Lys Leu Thr Asn Ser Ser His Arg Arg Lys Arg Ser Ile Gly Gln 530 535 540 Asn Val Thr Ser Cys Pro Tyr Val Ser Tyr Gly Arg Phe Cys Ile Glu 545 550 555 560 Pro Asp Gly Ser Leu Lys Met Ile Val Pro Glu Glu Leu Lys Gln Phe 565 570 575 Val Ala Pro Leu Leu Asn Ile Thr Glu Ser Val Leu Ile Pro Asn Ser 580 585 590 Phe Asn Leu Thr Val Thr Asp Glu Tyr Ile Gln Thr Arg Met Asp Lys 595 600 605 Val Gln Ile Asn Cys Leu Gln Tyr Val Cys Gly Asn Ser Leu Glu Cys 610 615 620 Arg Lys Leu Phe Gln Gln Tyr Gly Pro Val Cys Asp Asn Ile Leu Ser 625 630 635 640 Val Val Asn Ser Val Ser Gln Lys Glu Asp Met Glu Leu Leu Ser Phe 645 650 655 Tyr Ser Ser Thr Lys Pro Lys Gly Tyr Asp Thr Pro Val Leu Ser Asn 660 665 670 Val Ser Thr Gly Glu Phe Asn Ile Ser Leu Leu Leu Lys Pro Pro Ser 675 680 685 Ser Pro Ser Gly Arg Ser Phe Ile Glu Glu Leu Leu Phe Thr Ser Val 690 695 700 Glu Thr Val Gly Leu Pro Thr Asp Ala Glu Tyr Lys Lys Cys Thr Ala 705 710 715 720 Gly Pro Leu Gly Thr Leu Lys Asp Leu Ile Cys Ala Arg Glu Tyr Asn 725 730 735 Gly Leu Leu Val Leu Pro Pro Ile Ile Thr Ala Asp Met Gln Thr Met 740 745 750 Tyr Thr Ala Ser Leu Val Gly Ala Met Ala Phe Gly Gly Ile Thr Ser 755 760 765 Ala Ala Ala Ile Pro Phe Ala Thr Gln Ile Gln Ala Arg Ile Asn His 770 775 780 Leu Gly Ile Thr Gln Ser Leu Leu Met Lys Asn Gln Glu Lys Ile Ala 785 790 795 800 Ala Ser Phe Asn Lys Ala Ile Gly His Met Gln Glu Gly Phe Arg Ser 805 810 815 Thr Ser Leu Ala Leu Gln Gln Ile Gln Asp Val Val Asn Lys Gln Ser 820 825 830 Ala Ile Leu Thr Glu Thr Met Asn Ser Leu Asn Lys Asn Phe Gly Ala 835 840 845 Ile Thr Ser Val Ile Gln Asp Ile Tyr Ala Gln Leu Asp Ala Ile Gln 850 855 860 Ala Asp Ala His Val Asp Arg Leu Ile Thr Gly Arg Leu Ser Ser Leu 865 870 875 880 Ser Val Leu Ala Ser Ala Lys Gln Ser Glu Tyr Ile Arg Val Ser Gln 885 890 895 Gln Arg Glu Leu Ala Thr Gln Lys Ile Asn Glu Cys Val Lys Ser Gln 900 905 910 Ser Asn Arg Tyr Gly Phe Cys Gly Ser Gly Arg His Val Leu Ser Ile 915 920 925 Pro Gln Asn Ala Pro Asn Gly Ile Val Phe Ile His Phe Thr Tyr Thr 930 935 940 Pro Glu Ser Phe Val Asn Val Thr Ala Ile Val Gly Phe Cys Val Asn 945 950 955 960 Pro Ala Asn Ala Ser Gln Tyr Ala Ile Val Pro Ala Asn Gly Arg Gly 965 970 975 Ile Phe Ile Gln Val Asn Gly Thr Tyr Tyr Ile Thr Ala Arg Asp Met 980 985 990 Tyr Met Pro Arg Asp Ile Thr Ala Gly Asp Ile Val Thr Leu Thr Ser 995 1000 1005 Cys Gln Ala Asn Tyr Val Asn Val Asn Lys Thr Val Ile Thr Thr 1010 1015 1020 Phe Val Glu Asp Asp Asp Phe Asp Phe Asp Asp Glu Leu Ser Lys 1025 1030 1035 Trp Trp Asn Asp Thr Lys His Gln Leu Pro Asp Phe Asp Asp Phe 1040 1045 1050 Asn Tyr Thr Val Pro Ile Leu Asn Ile Ser Gly Glu Ile Asp Tyr 1055 1060 1065 Ile Gln Gly Val Ile Gln Gly Leu Asn Asp Ser Leu Ile Asp Leu 1070 1075 1080 Glu Glu Leu Ser Ile Ile Lys Thr Tyr Ile Lys Trp Pro Trp Tyr 1085 1090 1095 Val Trp Leu Ala Ile Phe Phe Ala Ile Ile Ile Phe Ile Leu Ile 1100 1105 1110 Leu Gly Trp Val Phe Phe Met Thr Gly Cys Cys Gly Cys Cys Cys 1115 1120 1125 Gly Cys Phe Gly Ile Ile Pro Leu Met Ser Lys Cys Gly Lys Lys 1130 1135 1140 Ser Ser Tyr Tyr Thr Thr Phe Asp Asn Asp Val Val Thr 1145 1150 1155 <210> 5 <211> 1156 <212> PRT <213> Avian infectious bronchitis virus <400> 5 Met Leu Gly Lys Ser Leu Phe Val Val Thr Leu Leu Phe Ala Leu Cys 1 5 10 15 Ser Ala Ala Leu Phe Asp Asn Asn Glu Thr Val Tyr Tyr Tyr Gln Ser 20 25 30 Ala Phe Arg Pro Ala Asp Gly Trp His Leu His Gly Gly Ala Tyr Ala 35 40 45 Val Val Asn Val Ser Leu Gln Thr Ser Asn Ala Gly Thr Val Ser Glu 50 55 60 Cys Ile Ala Gly Ala Ile Ser Trp Ser Lys Glu Phe Ser Ala Ser Ala 65 70 75 80 Val Ala Met Thr Ala Pro Gln Leu Gly Met Thr Trp Ser Thr Arg Gln 85 90 95 Phe Cys Thr Ala His Cys Asn Phe Ser Asp Phe Thr Val Phe Val Thr 100 105 110 His Cys Phe Lys His Gly Thr Gly Leu Cys Pro Leu Thr Gly Phe Ile 115 120 125 Pro Ser Gly Phe Ile Arg Val Ser Ala Met Arg Lys Gly Ser Asn Ser 130 135 140 Leu Phe Tyr Asn Leu Thr Val Ser Val Thr Lys Tyr Pro Arg Phe Lys 145 150 155 160 Ser Leu Gln Cys Val Asn Asn Tyr Thr Ser Val Tyr Leu Asn Gly Asp 165 170 175 Leu Val Phe Thr Ser Asn Glu Thr Lys Pro Val Ser Ala Ala Gly Val 180 185 190 Ser Phe Lys Ala Gly Gly Pro Ile Thr Tyr Lys Ile Met Asn Glu Val 195 200 205 Lys Val Leu Ala Tyr Phe Val Asn Gly Thr Ala Gln Thr Val Ile Pro 210 215 220 Cys Asp Gly Ser Pro Arg Gly Leu Leu Ala Cys Gln Tyr Asn Thr Gly 225 230 235 240 Asn Phe Ser Asp Gly Phe Tyr Pro Tyr Thr Asn Ser Ser Leu Val Lys 245 250 255 Glu Arg Phe Ile Val Tyr Arg Glu Ser Ser Val Asn Thr Ile Leu Val 260 265 270 Leu Thr Asn Phe Thr Phe Ser Asn Val Ser Asn Ala Leu Pro Asn Thr 275 280 285 Gly Asn Val His Ser Ile Val Leu His Gln Thr Gln Thr Ala Gln Ser 290 295 300 Gly Tyr Tyr Asn Leu Asn Phe Ser Phe Leu Ser Gly Phe Arg Tyr Val 305 310 315 320 Glu Ser Asp Phe Met Tyr Gly Ser Tyr His Pro Lys Cys Ser Phe Arg 325 330 335 Pro Glu Thr Ile Asn Asn Gly Leu Trp Phe Asn Ser Leu Ser Val Ser 340 345 350 Leu Gly Tyr Gly Pro Leu Gln Gly Gly Cys Lys Gln Ser Val Phe Asn 355 360 365 Asn Lys Ala Thr Cys Cys Tyr Ala Tyr Ser Tyr Asn Gly Pro Thr Leu 370 375 380 Cys Lys Gly Val Tyr Ser Gly Glu Leu Gln Lys Thr Phe Glu Cys Gly 385 390 395 400 Leu Leu Val Phe Val Thr Lys Ser Asp Gly Ser Arg Ile Gln Thr Arg 405 410 415 Asn Glu Pro Leu Val Leu Thr Gln His Asn Tyr Asn Asn Ile Thr Leu 420 425 430 Asn Lys Cys Val Glu Tyr Asn Ile Tyr Gly Arg Val Gly Gln Gly Leu 435 440 445 Ile Thr Asn Ile Thr Asp Ser Ala Ala Asn His Gly Tyr Leu Ala Asp 450 455 460 Gly Gly Leu Ala Val Leu Asp Thr Ser Gly Ala Ile Asp Val Phe Val 465 470 475 480 Val Gln Gly Val Tyr Gly Leu Asn Tyr Tyr Lys Val Asn Pro Cys Glu 485 490 495 Asp Val Asn Gln Gln Phe Val Val Ser Gly Gly Gln Leu Val Gly Ile 500 505 510 Leu Thr Ser Arg Asn Glu Thr Gly Ser Gln Pro Ile Glu Asn Arg Phe 515 520 525 Tyr Val Lys Phe Ser Asn Ser Arg Arg Arg Thr Gly Arg Ser Thr Ile 530 535 540 Ala Asn Val Thr Asn Cys Pro Tyr Val Ser Tyr Gly Lys Phe Cys Ile 545 550 555 560 Lys Pro Asp Gly Ser Val Leu Glu Ile Val Pro Gln Glu Ile Glu His 565 570 575 Phe Val Ala Pro Leu Leu Asn Val Thr Glu His Val Leu Ile Pro Asn 580 585 590 Ser Phe Asn Leu Thr Val Thr Asp Glu Tyr Ile Gln Thr Arg Met Asp 595 600 605 Lys Ile Gln Ile Asn Cys Leu Gln Tyr Val Cys Gly Asn Ser Ile Glu 610 615 620 Cys Arg Lys Leu Phe Gln Gln Tyr Gly Pro Val Cys Asp Asn Ile Leu 625 630 635 640 Ser Val Val Asn Thr Val Gly Gln Arg Glu Asp Met Glu Leu Leu Ser 645 650 655 Phe Tyr Ser Ser Thr Lys Pro Lys Asp Tyr Asn Val Pro Ile Phe Ser 660 665 670 Asn Val Ser Thr Gly Asp Phe Asn Ile Ser Leu Leu Leu Thr Pro Pro 675 680 685 Asn Ser Pro Thr Gly Arg Ser Phe Ile Glu Asp Ile Leu Phe Thr Ser 690 695 700 Val Glu Ser Val Gly Leu Pro Thr Asp Glu Glu Tyr Lys Lys Cys Thr 705 710 715 720 Ala Gly Pro Leu Gly Phe Val Lys Asp Leu Val Cys Ala Arg Glu Tyr 725 730 735 Asn Gly Leu Leu Val Leu Pro Pro Ile Ile Thr Ala Asp Met Gln Thr 740 745 750 Met Tyr Thr Ser Thr Leu Val Ala Ser Met Ala Leu Gly Gly Ile Thr 755 760 765 Ala Ala Gly Ala Ile Pro Phe Ala Thr Gln Leu Gln Ala Arg Ile Asn 770 775 780 His Leu Gly Ile Thr Gln Ser Leu Leu Leu Lys Asn Gln Glu Lys Ile 785 790 795 800 Ala Ala Ser Phe Asn Lys Ala Ile Gly His Met Gln Glu Gly Phe Arg 805 810 815 Ser Thr Ser Leu Ala Leu Gln Gln Ile Gln Asp Val Val Asn Lys Gln 820 825 830 Ser Ser Ile Leu Thr Glu Thr Met Ala Ser Leu Asn Lys Asn Phe Gly 835 840 845 Ala Ile Ser Ser Val Leu Gln Asp Ile Tyr Gln Gln Leu Asp Ala Ile 850 855 860 Gln Ala Asp Ala His Val Asp Arg Ile Ile Thr Gly Arg Leu Ser Ser 865 870 875 880 Leu Ser Val Leu Ala Ser Ala Lys Gln Ser Glu Tyr Tyr Arg Val Ser 885 890 895 Gln Gln Arg Glu Leu Ala Thr Gln Lys Ile Asn Glu Cys Val Lys Ser 900 905 910 Gln Ser Thr Arg Tyr Ser Phe Cys Gly Asn Gly Arg His Val Leu Thr 915 920 925 Ile Pro Gln Asn Ala Pro Asn Gly Ile Val Phe Ile His Phe Thr Tyr 930 935 940 Thr Pro Glu Ser Phe Val Asn Val Thr Ala Ile Val Gly Phe Cys Val 945 950 955 960 Asn Pro Pro Asn Ala Ser Gln Tyr Ala Leu Val Pro Ala Asn Gly Arg 965 970 975 Gly Ile Phe Ile Gln Val Asn Gly Ser Tyr Tyr Ile Thr Ala Arg Asp 980 985 990 Met Tyr Met Pro Arg Asp Ile Thr Ala Gly Asp Ile Val Thr Leu Thr 995 1000 1005 Ser Cys Gln Ala Asn Tyr Val Ser Val Asn Arg Thr Val Ile Thr 1010 1015 1020 Thr Phe Val Asp Asn Asp Asp Phe Asp Phe Asp Asp Glu Leu Ser 1025 1030 1035 Lys Trp Trp Asn Asp Thr Lys His Glu Leu Pro Asp Phe Asp Glu 1040 1045 1050 Phe Asn Tyr Thr Ile Pro Val Leu Asn Ile Ser Asn Glu Ile Asp 1055 1060 1065 Ile Ile Gln Glu Val Ile Arg Gly Leu Asn Asp Ser Leu Ile Asp 1070 1075 1080 Leu Glu Ala Leu Ser Ile Leu Lys Thr Tyr Ile Lys Trp Pro Trp 1085 1090 1095 Tyr Val Trp Leu Ala Ile Ala Phe Leu Thr Ile Ile Phe Ile Leu 1100 1105 1110 Val Leu Cys Trp Ile Phe Phe Met Thr Gly Cys Cys Gly Cys Cys 1115 1120 1125 Cys Gly Cys Phe Gly Ile Met Pro Leu Met Ser Lys Cys Gly Lys 1130 1135 1140 Lys Ser Ser Tyr Tyr Thr Thr Phe Asp Asn Asp Val Val 1145 1150 1155 <210> 6 <211> 1156 <212> PRT <213> Avian infectious bronchitis virus <400> 6 Met Leu Val Lys Ser Leu Phe Ile Val Thr Leu Leu Phe Ala Leu Cys 1 5 10 15 Ser Ala Ala Leu Phe Asp Asn Asn Gln Ala Val Tyr Tyr Tyr Gln Ser 20 25 30 Ala Phe Arg Pro Ser Ser Gly Trp His Lys His Gly Gly Ala Tyr Ala 35 40 45 Val Ala Asn Val Ser Leu Glu Tyr Ala Asn Ala Gly Ser Ser Thr His 50 55 60 Cys Thr Ala Gly Ala Ile Tyr Trp Ser Lys Asn Phe Thr Ala Ser Ser 65 70 75 80 Val Ala Met Thr Ala Pro Gly Thr Gly Met Ser Trp Ser Thr Ala Gln 85 90 95 Phe Cys Thr Ala His Cys Asn Phe Ser Asp Phe Thr Val Phe Val Thr 100 105 110 His Cys Tyr Lys Ser Gly Asp Val Cys Pro Leu Thr Gly Leu Ile Pro 115 120 125 Ser Gly Tyr Ile Arg Ile Ser Ala Met Thr Lys Gly Thr Thr Ser Leu 130 135 140 Phe Tyr Asn Leu Thr Val Pro Val Thr Lys Tyr Pro Lys Phe Lys Ser 145 150 155 160 Leu Gln Cys Val Asp Asn Phe Thr Ser Val Tyr Leu Asn Gly Asp Leu 165 170 175 Val Phe Thr Ser Asn Glu Thr Lys Asp Val Ser Ala Ala Gly Val His 180 185 190 Phe Lys Ala Gly Gly Pro Ile Thr Tyr Lys Val Met Glu Lys Val Asp 195 200 205 Val Leu Ala Tyr Phe Val Asn Gly Thr Ala Gln Asp Val Ile Leu Cys 210 215 220 Asp Asn Ser Pro Arg Gly Leu Leu Ala Cys Gln Tyr Asn Thr Gly Asn 225 230 235 240 Phe Ser Asp Gly Phe Tyr Pro Phe Thr Asn Ile Ser Leu Val Lys Glu 245 250 255 Lys Phe Ile Val Tyr Arg Glu Thr Ser Val Asn Thr Thr Leu Val Leu 260 265 270 Thr Asn Phe Thr Phe Thr Asn Val Ser Asn Ala Leu Pro Asn Thr Gly 275 280 285 Gly Val Asn Thr Ile Asn Ile Tyr Gln Thr Gln Thr Ala Gln Ser Gly 290 295 300 Cys Tyr Asn Phe Asn Phe Ser Phe Leu Ser Ser Phe Val Tyr Lys Gln 305 310 315 320 Ser Asp Phe Met Tyr Gly Ser Tyr His Pro Lys Cys Asp Phe Arg Pro 325 330 335 Glu Thr Ile Asn Asn Gly Leu Trp Phe Asn Ser Leu Ser Val Ser Leu 340 345 350 Ala Tyr Gly Pro Leu Gln Gly Gly Cys Lys Gln Ser Val Phe Ser Asn 355 360 365 Arg Ala Thr Cys Cys Tyr Ala Tyr Ser Tyr Asn Gly Pro Arg Leu Cys 370 375 380 Lys Gly Val Tyr Ile Gly Glu Leu Gln Gln Tyr Phe Glu Cys Gly Leu 385 390 395 400 Leu Val Tyr Val Thr Lys Ser Asp Gly Ser Arg Ile Gln Thr Arg Asn 405 410 415 Glu Pro Leu Val Leu Thr His His Asn Tyr Asn Asn Ile Thr Leu Asp 420 425 430 Arg Cys Val Glu Tyr Asn Ile Tyr Gly Arg Ser Gly Gln Gly Phe Ile 435 440 445 Thr Asn Val Thr Ala Ala Ala Ala Asn Tyr Asn Tyr Leu Ala Asp Gly 450 455 460 Gly Leu Ala Ile Leu Asp Thr Ser Gly Ala Ile Asp Ile Phe Val Val 465 470 475 480 Gln Gly Glu Tyr Gly Pro Asn Tyr Tyr Lys Val Asn Pro Cys Glu Asp 485 490 495 Val Asn Gln Gln Phe Val Val Ser Gly Gly Gly Ile Val Gly Val Leu 500 505 510 Thr Ser His Asn Glu Thr Gly Ser Gln Gln Leu Glu Asn Leu Phe Tyr 515 520 525 Val Lys Leu Thr Asn Ser Thr Arg Arg Thr Arg Arg Ser Thr Ile Ala 530 535 540 Asn Val Thr Thr Cys Pro Tyr Val Ser Tyr Gly Arg Phe Cys Ile Lys 545 550 555 560 Pro Asp Gly Leu Val Ser Glu Ile Val Pro Gln Glu Leu Asp Tyr Phe 565 570 575 Val Ala Pro Leu Leu Asn Val Thr Glu His Val Leu Ile Pro Asn Ser 580 585 590 Phe Asn Leu Thr Val Thr Asp Glu Tyr Ile Gln Thr Arg Met Glu Lys 595 600 605 Val Gln Ile Asn Cys Leu Gln Tyr Val Cys Gly Asn Ser Ile Glu Cys 610 615 620 Arg Asn Leu Phe Gln Gln Tyr Gly Pro Val Cys Asp Asn Ile Leu Ser 625 630 635 640 Ile Val Asn Ser Val Gly Gln Arg Glu Asp Met Glu Ser Leu Thr Phe 645 650 655 Tyr Ser Ser Thr Lys Pro Lys Gly Tyr Asn Thr Pro Ile Phe Ser Asn 660 665 670 Ile Ser Thr Gly Asp Phe Asn Ile Ser Leu Met Leu Thr Pro Pro Ser 675 680 685 Ser Pro Ser Gly Arg Ser Phe Ile Glu Asp Leu Leu Phe Thr Ser Val 690 695 700 Glu Thr Val Gly Leu Pro Thr Asp Ala Glu Tyr Lys Lys Cys Thr Ala 705 710 715 720 Gly Pro Leu Gly Thr Leu Lys Asp Leu Ile Cys Ala Arg Glu Tyr Asn 725 730 735 Gly Leu Leu Val Leu Pro Pro Ile Ile Thr Ala Asp Met Gln Thr Met 740 745 750 Tyr Thr Ala Ser Leu Val Gly Ala Met Ala Phe Gly Gly Ile Thr Ser 755 760 765 Ala Ala Ala Ile Pro Phe Ala Thr Gln Ile Gln Ala Arg Ile Asn His 770 775 780 Leu Gly Ile Thr Gln Ser Leu Leu Met Lys Asn Gln Glu Lys Ile Ala 785 790 795 800 Ala Ser Phe Asn Lys Ala Ile Gly His Met Gln Glu Gly Phe Arg Ser 805 810 815 Thr Ser Leu Ala Leu Gln Gln Ile Gln Asp Val Val Asn Lys Gln Ser 820 825 830 Ala Ile Leu Thr Glu Thr Met Asn Ser Leu Asn Lys Asn Phe Gly Ala 835 840 845 Ile Thr Ser Val Ile Gln Asp Ile Tyr Ala Gln Leu Asp Ala Ile Gln 850 855 860 Ala Asp Ala His Val Asp Arg Leu Ile Thr Gly Arg Leu Ser Ser Leu 865 870 875 880 Ser Val Leu Ala Ser Ala Lys Gln Ser Glu Tyr Ile Arg Val Ser Gln 885 890 895 Gln Arg Glu Leu Ala Thr Gln Lys Ile Asn Glu Cys Val Lys Ser Gln 900 905 910 Ser Asn Arg Tyr Gly Phe Cys Gly Ser Gly Arg His Val Leu Ser Ile 915 920 925 Pro Gln Asn Ala Pro Asn Gly Ile Val Phe Ile His Phe Thr Tyr Thr 930 935 940 Pro Glu Ser Phe Val Asn Val Thr Ala Ile Val Gly Phe Cys Val Ser 945 950 955 960 Pro Ala Asn Ala Ser Gln Tyr Ala Ile Val Pro Ala Asn Gly Arg Gly 965 970 975 Ile Phe Ile Gln Val Asn Gly Thr Tyr Tyr Ile Thr Ala Arg Asp Met 980 985 990 Tyr Met Pro Arg Asp Ile Thr Ala Gly Asp Ile Val Thr Leu Thr Ser 995 1000 1005 Cys Gln Ala Asn Tyr Val Asn Val Asn Lys Thr Val Ile Thr Thr 1010 1015 1020 Phe Val Glu Asp Asp Asp Phe Asp Phe Asp Asp Glu Leu Ser Lys 1025 1030 1035 Trp Trp Asn Glu Thr Lys His Glu Ile Pro Asp Phe Asp Glu Phe 1040 1045 1050 Asn Tyr Thr Val Pro Ile Leu Asn Ile Ser Ser Glu Ile Asp Arg 1055 1060 1065 Ile Gln Gly Val Ile Gln Gly Leu Asn Asp Ser Leu Ile Asn Leu 1070 1075 1080 Glu Glu Leu Ser Ile Ile Lys Thr Tyr Ile Lys Trp Pro Trp Tyr 1085 1090 1095 Val Trp Leu Ala Ile Gly Phe Ala Ile Ile Ile Phe Ile Leu Ile 1100 1105 1110 Leu Gly Trp Val Phe Phe Met Thr Gly Cys Cys Gly Cys Cys Cys 1115 1120 1125 Gly Cys Phe Gly Ile Ile Pro Leu Met Ser Lys Cys Gly Lys Lys 1130 1135 1140 Ser Ser Tyr Tyr Thr Thr Phe Asp Asn Asp Val Val Thr 1145 1150 1155 <210> 7 <211> 1169 <212> PRT <213> Avian infectious bronchitis virus <400> 7 Met Leu Val Gln Pro Leu Leu Leu Val Thr Leu Leu Cys Ala Leu Cys 1 5 10 15 Ser Ala Ser Leu Tyr Asn Asn Asp Ser Tyr Val Tyr Tyr Tyr Gln Ser 20 25 30 Ala Phe Arg Pro Phe Asn Gly Trp His Leu His Gly Gly Ala Tyr Ala 35 40 45 Val Val Asn Val Ser Gln Glu Thr Ala Asn Ala Gly Ser Ser Pro Ser 50 55 60 Cys Thr Ala Gly Ala Ile Tyr Trp Ser Lys Asn Phe Thr Ala Ser Ser 65 70 75 80 Val Ala Met Thr Ala Pro Leu Gln Gly Met Gln Trp Ser Thr Ile Gln 85 90 95 Phe Cys Thr Ala His Cys Asn Phe Thr Asn Ile Val Val Phe Val Thr 100 105 110 His Cys Tyr Lys Ser Gly Ser Thr Val Cys Pro Leu Thr Gly Leu Ile 115 120 125 Pro Gln Asn His Ile Arg Ile Ser Ala Met Lys Gln Gly Asn Asn Gly 130 135 140 Pro Ser Gly Leu Phe Tyr Asn Leu Thr Val Pro Val Thr Lys Tyr Ser 145 150 155 160 Lys Phe Lys Ser Leu Gln Cys Val Asn Asn Gln Thr Ser Val Tyr Leu 165 170 175 Asn Gly Asp Leu Val Phe Thr Ser Asn Glu Thr Lys Asp Val Ser Gly 180 185 190 Ala Gly Val Tyr Phe Lys Ala Gly Gly Pro Ile Thr Tyr Lys Val Met 195 200 205 Arg Glu Val Lys Ala Leu Ala Tyr Phe Val Asn Gly Thr Ala His Asp 210 215 220 Val Ile Leu Cys Asp Gly Ser Pro Arg Gly Leu Leu Ala Cys Gln Tyr 225 230 235 240 Asn Thr Gly Lys Phe Ser Asp Gly Phe Tyr Pro Phe Thr Asn Asp Thr 245 250 255 Leu Val Lys Glu Lys Phe Ile Val Tyr Arg Glu Asn Ser Val Asn Thr 260 265 270 Thr Leu Thr Leu Thr Asn Phe Thr Phe Tyr Asn Glu Ser Asn Ala Leu 275 280 285 Pro Asn Asn Gly Gly Val Asp Thr Ile Gln Leu Tyr Gln Thr His Thr 290 295 300 Ala Gln Ser Gly Tyr Tyr Asn Phe Asn Phe Ser Phe Leu Ser Ser Phe 305 310 315 320 Gln Tyr Val Glu Ser Asn Phe Met Tyr Gly Ser Tyr His Pro Lys Cys 325 330 335 Gly Phe Arg Pro Glu Ser Ile Asn Asn Gly Leu Trp Phe Asn Ser Leu 340 345 350 Ser Val Ser Leu Ala Tyr Gly Pro Leu Gln Gly Gly Cys Lys Gln Ser 355 360 365 Val Phe His Gly Arg Ala Thr Cys Cys Tyr Ala Tyr Ser Tyr Asn Gly 370 375 380 Pro Thr Leu Cys Lys Gly Val Tyr Ser Gly Glu Leu Thr Arg Ser Tyr 385 390 395 400 Gln Cys Gly Leu Leu Val Phe Val Thr Lys Ser Asp Gly Ser Arg Ile 405 410 415 Gln Thr Ser Thr Lys Pro Ile Val Leu Thr Gln His Asn Tyr Asn Asn 420 425 430 Ile Thr Leu Asp Arg Cys Val Glu Tyr Asn Ile Tyr Gly Arg Val Gly 435 440 445 Gln Gly Phe Ile Thr Asn Val Thr Glu Ser Ala Ala Ala Phe Asn Tyr 450 455 460 Leu Glu Asp Gly Gly Leu Ala Ile Leu Asp Thr Ser Gly Ala Ile Asp 465 470 475 480 Ile Phe Val Val Gln Gly Glu Tyr Gly Phe Asn Tyr Tyr Lys Val Asn 485 490 495 Pro Cys Glu Asp Val Asn Gln Gln Phe Val Val Ser Gly Gly Asn Leu 500 505 510 Val Gly Ile Leu Thr Ser Ile Asn Gln Thr Gly Ser Gln Ser Ile Glu 515 520 525 Asn Gln Phe Tyr Val Lys Leu Thr Asn Gly Ser Arg Arg Ser Arg Arg 530 535 540 Ser Val Ser Glu Asn Val Thr Ser Cys Pro Tyr Val Ser Tyr Gly Lys 545 550 555 560 Phe Cys Ile Lys Pro Asp Gly Ser Leu Ser Thr Ile Val Pro Lys Glu 565 570 575 Leu Glu Gln Phe Val Ala Pro Leu Leu Asn Val Thr Glu His Val Leu 580 585 590 Ile Pro Asp Ser Phe Asn Leu Thr Val Thr Asp Glu Tyr Ile Gln Thr 595 600 605 Arg Met Asp Lys Val Gln Ile Asn Cys Leu Gln Tyr Val Cys Gly Asn 610 615 620 Ser Phe Glu Cys Arg Lys Leu Phe Gln Gln Tyr Gly Pro Val Cys Asp 625 630 635 640 Asn Ile Leu Ser Val Val Asn Ser Val Gly Gln Lys Glu Asp Met Glu 645 650 655 Leu Leu Ser Phe Tyr Ser Ser Thr Lys Pro Ser Gly Ile Ser Gln Pro 660 665 670 Leu Phe Asn Asn Phe Ser Thr Gly Asp Phe Asn Ile Ser Leu Leu Leu 675 680 685 Thr Ser Pro Ser Ser Pro Ser Gly Arg Ser Phe Ile Glu Asp Leu Leu 690 695 700 Phe Thr Ser Val Glu Ser Val Gly Leu Pro Thr Asp Glu Ala Tyr Lys 705 710 715 720 Lys Cys Thr Ser Gly Pro Leu Gly Phe Val Lys Asp Leu Val Cys Ala 725 730 735 Arg Glu Tyr Asn Gly Leu Leu Val Leu Pro Pro Ile Ile Thr Ala Glu 740 745 750 Met Gln Thr Met Tyr Thr Ser Ser Leu Val Ala Ser Met Ala Leu Gly 755 760 765 Gly Ile Thr Ala Ala Gly Ala Ile Pro Phe Ala Thr Gln Leu Gln Ala 770 775 780 Arg Ile Asn His Leu Gly Ile Thr Gln Ser Leu Leu Met Lys Asn Gln 785 790 795 800 Glu Lys Ile Ala Ala Ser Phe Asn Arg Ala Ile Gly His Met Gln Glu 805 810 815 Gly Phe Lys Ser Thr Ser Leu Ala Leu Gln Gln Ile Gln Asp Val Val 820 825 830 Asn Lys Gln Ser Ala Ile Leu Thr Glu Thr Met Ala Ser Leu Asn Lys 835 840 845 Asn Phe Gly Ala Ile Ser Ser Val Leu Gln Asp Ile Tyr Gln Gln Leu 850 855 860 Asp Val Ile Gln Ala Asp Ala His Val Asp Arg Ile Ile Thr Gly Arg 865 870 875 880 Leu Ser Ser Leu Ser Val Leu Ala Ser Ala Lys Gln Ser Glu Tyr Ile 885 890 895 Ala Val Ser Gln Gln Arg Ala Leu Ala Thr Gln Lys Ile Asn Glu Cys 900 905 910 Val Lys Ser Gln Ser Thr Arg Tyr Ser Phe Cys Gly Asn Gly Arg His 915 920 925 Val Leu Thr Ile Pro Gln Asn Ala Pro Asn Gly Ile Val Phe Ile His 930 935 940 Phe Thr Tyr Thr Pro Glu Ser Phe Val Asn Val Thr Ala Ile Val Gly 945 950 955 960 Phe Cys Val Lys Pro Pro Asn Ala Ser His Tyr Ala Ile Val Pro Val 965 970 975 Asn Gly Arg Gly Ile Phe Ile Gln Val Asn Gly Thr Tyr Tyr Ile Thr 980 985 990 Ser Arg Asp Met Tyr Met Pro Arg Asn Ile Thr Ala Gly Asp Ile Val 995 1000 1005 Thr Leu Thr Ser Cys Gln Ala Asn Tyr Val Ser Val Asn Lys Thr 1010 1015 1020 Val Ile Ser Thr Phe Val Glu Asp Asp Asp Phe Asp Phe Asp Asp 1025 1030 1035 Glu Leu Ser Lys Trp Trp Asn Asp Thr Lys His Glu Leu Pro Asp 1040 1045 1050 Phe Asp Glu Phe Asn Tyr Thr Ile Pro Val Leu Asn Ile Ser Asn 1055 1060 1065 Glu Ile Asp Arg Ile Gln Gly Val Ile Gln Gly Leu Asn Asp Ser 1070 1075 1080 Leu Ile Asp Leu Glu Thr Leu Ser Ile Leu Lys Thr Tyr Ile Lys 1085 1090 1095 Trp Pro Trp Tyr Val Trp Leu Ala Ile Phe Phe Ala Ile Val Ile 1100 1105 1110 Phe Ile Leu Ile Ile Gly Trp Val Phe Phe Met Thr Gly Cys Cys 1115 1120 1125 Gly Cys Cys Cys Gly Cys Phe Gly Ile Ile Pro Leu Met Asn Lys 1130 1135 1140 Cys Gly Lys Lys Ser Ser Tyr Tyr Thr Thr Phe Asp Asn Asp Val 1145 1150 1155 Val Thr Glu Gln Tyr Arg Pro Lys Lys Ser Val 1160 1165 <210> 8 <211> 1159 <212> PRT <213> Avian infectious bronchitis virus <400> 8 Met Leu Val Lys Ser Leu Phe Leu Val Thr Ile Leu Phe Ala Leu Cys 1 5 10 15 Ser Ala Asn Leu Tyr Asp Asn Glu Ser Phe Val Tyr Tyr Tyr Gln Ser 20 25 30 Ala Phe Arg Pro Gly His Gly Trp His Leu His Gly Gly Ala Tyr Ala 35 40 45 Val Val Asn Val Ser Ser Glu Asn Asn Asn Ala Gly Thr Ala Pro Ser 50 .55 60 Cys Thr Ala Gly Ala Ile Gly Tyr Ser Lys Asn Phe Ser Ala Ala Ser 65 70 75 80 Val Ala Met Thr Ala Pro Leu Ser Gly Met Ser Trp Ser Ala Ser Ser 85 90 95 Phe Cys Thr Ala His Cys Asn Phe Thr Ser Tyr Ile Val Phe Val Thr 100 105 110 His Cys Phe Lys Ser Gly Ser Asn Ser Cys Pro Leu Thr Gly Leu Ile 115 120 125 Pro Ser Gly Tyr Ile Arg Ile Ala Ala Met Lys His Gly Ser Arg Thr 130 135 140 Pro Gly His Leu Phe Tyr Asn Leu Thr Val Ser Val Thr Lys Tyr Pro 145 150 155 160 Lys Phe Arg Ser Leu Gln Cys Val Asn Asn His Thr Ser Val Tyr Leu 165 170 175 Asn Gly Asp Leu Val Phe Thr Ser Gly Tyr Thr Glu Asp Val Val Ala 180 185 190 Ala Gly Val His Phe Lys Ser Gly Gly Pro Ile Thr Tyr Lys Val Met 195 200 205 Arg Glu Val Lys Ala Leu Ala Tyr Phe Val Asn Gly Thr Ala His Asp 210 215 220 Val Ile Leu Cys Asp Asp Thr Pro Arg Gly Leu Leu Ala Cys Gln Tyr 225 230 235 240 Asn Thr Gly Asn Phe Ser Asp Gly Phe Tyr Pro Phe Thr Asn Thr Ser 245 250 255 Ile Val Lys Asp Lys Phe Ile Val Tyr Arg Glu Ser Ser Val Asn Thr 260 265 270 Thr Leu Thr Leu Thr Asn Phe Thr Phe Ser Asn Glu Ser Gly Ala Pro 275 280 285 Pro Asn Thr Gly Gly Val Asp Ser Phe Ile Leu Tyr Gln Thr Gln Thr 290 295 300 Ala Gln Ser Gly Tyr Tyr Asn Phe Asn Phe Ser Phe Leu Ser Ser Phe 305 310 315 320 Val Tyr Arg Glu Ser Asn Tyr Met Tyr Gly Ser Tyr His Pro Arg Cys 325 330 335 Ser Phe Arg Pro Glu Thr Leu Asn Gly Leu Trp Phe Asn Ser Leu Ser 340 345 350 Val Ser Leu Thr Tyr Gly Pro Ile Gln Gly Gly Cys Lys Gln Ser Val 355 360 365 Phe Asn Gly Lys Ala Thr Cys Cys Tyr Ala Tyr Ser Tyr Gly Gly Pro 370 375 380 Arg Ala Cys Lys Gly Val Tyr Arg Gly Glu Leu Thr Gln His Phe Glu 385 390 395 400 Cys Gly Leu Leu Val Tyr Val Thr Lys Ser Asp Gly Ser Arg Ile Gln 405 410 415 Thr Ala Thr Gln Pro Pro Val Leu Thr Gln Asn Phe Tyr Asn Asn Ile 420 425 430 Thr Leu Gly Lys Cys Val Asp Tyr Asn Val Tyr Gly Arg Thr Gly Gln 435 440 445 Gly Phe Ile Thr Asn Val Thr Asp Leu Ala Thr Ser His Asn Tyr Leu 450 455 460 Ala Asp Gly Gly Leu Ala Ile Leu Asp Thr Ser Gly Ala Ile Asp Ile 465 470 475 480 Phe Val Val Gln Gly Glu Tyr Gly Pro Asn Tyr Tyr Lys Val Asn Leu 485 490 495 Cys Glu Asp Val Asn Gln Gln Phe Val Val Ser Gly Gly Lys Leu Val 500 505 510 Gly Ile Leu Thr Ser Arg Asn Glu Thr Gly Ser Gln Pro Leu Glu Asn 515 520 525 Gln Phe Tyr Ile Lys Ile Thr Asn Gly Thr His Arg Ser Arg Arg Ser 530 535 540 Val Asn Glu Asn Val Thr Asn Cys Pro Tyr Val Ser Tyr Gly Lys Phe 545 550 555 560 Cys Ile Lys Pro Asp Gly Ser Val Ser Pro Ile Val Pro Lys Glu Leu 565 570 575 Glu Gln Phe Val Ala Pro Leu Leu Asn Val Thr Glu Asn Val Leu Ile 580 585 590 Pro Asn Ser Phe Asn Leu Thr Val Thr Asp Glu Tyr Ile Gln Thr His 595 600 605 Met Asp Lys Ile Gln Ile Asn Cys Leu Gln Tyr Val Cys Gly Asn Ser 610 615 620 Leu Ala Cys Arg Lys Leu Phe Gln Gln Tyr Gly Pro Val Cys Asp Asn 625 630 635 640 Ile Leu Ser Val Val Asn Ser Val Gly Gln Lys Glu Asp Met Glu Leu 645 650 655 Leu Ser Phe Tyr Ser Ser Thr Lys Pro Ser Gly Phe Asn Thr Pro Val 660 665 670 Phe Ser Asn Leu Ser Thr Gly Glu Phe Asn Ile Ser Leu Leu Leu Thr 675 680 685 Thr Pro Ser Ser Pro Arg Gly Arg Ser Phe Ile Glu Asp Leu Leu Phe 690 695 700 Thr Ser Val Glu Ser Val Gly Leu Pro Thr Asp Glu Ala Tyr Lys Lys 705 710 715 720 Cys Thr Ala Gly Pro Leu Gly Phe Leu Lys Asp Leu Ala Cys Ala Arg 725 730 735 Glu Tyr Asn Gly Leu Leu Val Leu Pro Pro Ile Ile Thr Ala Glu Met 740 745 750 Gln Thr Leu Tyr Thr Ser Ser Leu Val Val Ser Met Ala Phe Gly Gly 755 760 765 Ile Thr Ser Ala Gly Ala Ile Pro Phe Ala Thr Gln Leu Gln Ala Arg 770 775 780 Ile Asn His Leu Gly Ile Thr Gln Ser Leu Leu Leu Lys Asn Gln Glu 785 790 795 800 Lys Ile Ala Ala Ser Phe Asn Lys Ala Ile Gly His Met Gln Glu Gly 805 810 815 Phe Arg Ser Thr Ser Leu Ala Leu Gln Gln Ile Gln Asp Val Val Asn 820 825 830 Lys Gln Ser Ala Ile Leu Thr Glu Thr Met Ala Ser Leu Asn Lys Asn 835 840 845 Phe Gly Ala Ile Ser Ser Val Ile Gln Glu Ile Tyr Gln Gln Leu Asp 850 855 860 Ala Ile Gln Ala Asn Ala His Val Asp Arg Leu Ile Thr Gly Arg Leu 865 870 875 880 Ser Ser Leu Ser Val Leu Ala Ser Ala Lys Gln Ala Glu Tyr Ile Arg 885 890 895 Val Ser Gln Gln Arg Glu Leu Ala Thr Gln Lys Ile Asn Glu Cys Val 900 905 910 Lys Ser Gln Ser Ile Arg Tyr Ser Phe Cys Gly Asn Gly Arg His Val 915 920 925 Leu Thr Ile Pro Gln Asn Ala Pro Asn Gly Ile Val Phe Ile His Phe 930 935 940 Ser Tyr Thr Pro Asp Ser Phe Val Asn Val Thr Ala Ile Val Gly Phe 945 950 955 960 Cys Val Lys Pro Ala Asn Ala Ser Gln Tyr Ala Ile Val Pro Ala Asn 965 970 975 Gly Arg Gly Ile Phe Ile Gln Val Asn Gly Ser Tyr Tyr Ile Thr Ala 980 985 990 Arg Asp Met Tyr Met Pro Arg Ala Ile Thr Ala Gly Asp Ile Val Thr 995 1000 1005 Leu Thr Ser Cys Gln Ala Asn Tyr Val Ser Val Asn Lys Thr Val 1010 1015 1020 Ile Thr Thr Phe Val Asp Asn Asp Asp Phe Asp Phe Asn Asp Glu 1025 1030 1035 Leu Ser Lys Trp Trp Asn Asp Thr Lys His Glu Leu Pro Asp Phe 1040 1045 1050 Asp Lys Phe Asn Tyr Thr Val Pro Ile Leu Asp Ile Asp Ser Glu 1055 1060 1065 Ile Asp Arg Ile Gln Gly Val Ile Gln Gly Leu Asn Asp Ser Leu 1070 1075 1080 Ile Asp Leu Glu Lys Leu Ser Ile Leu Lys Thr Tyr Ile Lys Trp 1085 1090 1095 Pro Trp Tyr Val Trp Leu Ala Ile Ala Phe Ala Thr Ile Ile Phe 1100 1105 1110 Ile Leu Ile Leu Gly Trp Val Phe Phe Met Thr Gly Cys Cys Gly 1115 1120 1125 Cys Cys Cys Gly Cys Phe Gly Ile Met Pro Leu Met Ser Lys Cys 1130 1135 1140 Gly Lys Lys Ser Ser Tyr Tyr Thr Thr Phe Asp Asn Asp Val Val 1145 1150 1155 Thr <210> 9 <211> 11398 <212> DNA <213> Artificial sequence <220> <223> Plasmid sequence <400> 9 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 240 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 300 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 360 tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt ggagatcggt acttcgcgaa 420 tgcgtcgaga tcggccgccc gggtaatacg actcactata gggacttaag atagatatta 480 atatatatct attgcactag ccttgcgcta gatttcaaac ttaacaaaac ggacttaaat 540 acctacagct ggtccccata ggtgttccat tgcagtgcac tttagtgccc tggatggcac 600 ctggccacct gtcaggtttt tgttgttaaa atatcattgt tgctggtatc actgcttgtt 660 ttgccgtgtc tcactttata catccgttgc ttgggctacc tagtatccag cgtcctacgg 720 gcgccgtggt cggttcgagt gcgaaggacc tctggttcat ctagcggtag gcgggtgtgt 780 ggaagtagcg cttcagacgt actggttctg ttgcgtgaaa cgcggggtca cctcccccca 840 cataccta agggctttg agcctagcgt tgggctacgt tctcgcacaa ggtcggctat 900 acgacgtttg tagggggtag tgccaaacaa cccctgaggt gacaggttct ggtggtgttt 960 cgaaaacaac aatgtgtgtg ccgcataata tgcgcgttat gcattttgga gcaggtagtg 1020 ataaaggagt ggcgcctggt agtactgttc ttaaacagtg gctccccgaa ggaacactcc 1080 ttgtcgataa tgatattgta gattatgttt ctgacgcaca cgtttctgtg ctttcagatt 1140 gcaataaata taagacagag cacaagtttg atcttgtgat atctgatatg tatacagaca 1200 atgattcaaa aagaaagcat gaaggcgtga tagccaataa tggcaatgat gacgtcttca 1260 tataccttc aagttttcta cgcaataatt tggctctggg aggcagtttt gctgtaaaat 1320 taacagagac aagttggcat gagagtttat atgacattgc acaggattgt gcatggtgga 1380 caatgttttg tacagcagtg aatgcatctt cttcagaagc attcctgatt ggtgttaatt 1440 acttgggtgc aagtgcaaag gttaaagtta gtggaaaaac actgcacgca aattatatat 1500 tttggaggaa ttgtaattat ttacaaacct ctgcttatag tatatttgat gttgctaagt 1560 ttgatttgag attgaaagca acgccagttg ttaatcttaa gactgaacaa aagacagact 1620 tagtctttaa tttgataaag tgtggtaagt tactggtaag agatgttggt aacctctt 1680 ttactagtga ctctttgtg tgcactatgt agtgctgctt tgtatgacag tagttcttac 1740 gtgtactact accaaagtgc cttcagacca cctgatggtt ggcatttaca tgggggtgcg 1800 tatgcggttg ttaatattc tagtgaatct aataatgcag gctcttcatc tgggtgtact 1860 gttggtatta ttcatggtgg tcgtgttgtt aatgcttctt ctatagctat gacggcaccg 1920 tcatcaggta tggcttggtc tagcagtcag ttttgtactg catactgtaa cttttcagat 1980 actacagtgt ttgttacaca ttgtataaa catggtgggt gtcctataac tggcatgctt 2040 caacagcatt ctatacgtgt ttctgctatg aaaaatggcc agcttttttta taatttaaca 2100 gttagtgtag ctaagtaccc tacttttaaa tcatttcagt gtgttaataa tttaacatcc 2160 gtatatttaa atggtgatct tgtttacacc tctaatgaga ccacagatgt tacatctgca 2220 ggtgtttatt ttaaagctgg tggacctata acttataaag ttatgagaga agttagagcc 2280 ctggcttatt ttgttaatgg tactgcacaa gatgttattt tgtgtgatgg gtcacctaga 2340 ggcttgttag catgccagta tatactggc aatttttcag atggctttta tccttttact 2400 aatagtagtt tagttaagca gaagtttatt gtctatcgtg aaaatagtgt tatactact 2460 tttacgttac acaatttcac ttttcataat gagactggcg ccaacccaaa tcctagtggt 2520 gtccagaata ttcaaactta ccaaacacaa acagctcaga gtggttatta taattttaat 2580 ttttccttc tgagtagttt tgtttataag gagtctaatt ttatgtatgg atcttatcac 2640 ccaagttgta atttagact agaaactatt aataatggtt tgtggtttaa ttcactttca 2700 gtttcaattg cttacggtcc tcttcaaggt ggttgcaagc aatctgtctt tagtggtaga 2760 gcaacctgtt gttatgctta ctcatatgga ggtcctttgc tgtgtaaagg tgtttattca 2820 ggtgagttag atcataattt tgaatgtgga ctgttagttt atgttactaa gagcggtggc 2880 tctcgtatac aaacagccac tgaaccgcca gttataactc aacacaatta tataatatt 2940 actttaaata cttgtgttga ttataatata tatggcagaa ctggccaagg ttttattact 3000 aatgtaaccg actcagctgt tagttaat tatctagcag acgcaggttt ggctatttta 3060 gatacatctg gttccataga catctttgtc gtacaaagtg aatatggtct taattattat 3120 aaggttaacc cttgcgaaga tgtcaaccag cagtttgtag tttctggtgg taaattagta 3180 ggtattctta cttcacgtaa tgagactggt tcccagcttc ttgagaatca gttttacatc 3240 aaaatcacta atggaacacg tcgttttaga cgttctatta ctgaaagtgt tgaaaattgc 3300 ccttatgtta gttatggtaa gttttgtata aaacctgatg gttcaattgc cacaatagta 3360 ccaaaacaat tggaacagtt tgtggcacct ttacttaatg ttactgaaaa tgtgctcata 3420 cctaacagtt ttaatttaac tgttacagat gagtacatac aaacgcgtat ggataaggtc 3480 caaattaatt gcctgcagta tatttgtggc aattctctgg agtgcagaaa tttgtttcaa 3540 caatatggtc ctgtttgcga caacatattg tctgtagtaa atagtgttgg tcaaaaagaa 3600 gatatggaac ttttgaattt ctattcttct actaagccgg ctggttttaa tacaccagtt 3660 cttagtaatg ttagcactgg tgagtttaat attactcttt ttttaacaac gcctagtagt 3720 cctagaaggc gttcttttat tgaagacctt ctatttacaa gtgttgaatc tgttggatta 3780 ccaacagatg acgcatacaa aaattgcact gcaggtcctt taggctttct taaggacctt 3840 gcatgtgctc gtgaatataa tggtttgctt gtgttgcctc ctattataac agcagaaatg 3900 caaactttgt atactagttc tctagtagct tctatggctt ttggtggtat tactgcagct 3960 ggtgctatac cttttgccac acaactgcag gctagaatta atcacttggg tattacccag 4020 tcacttcttt tgaagaatca agaaaaaatt gctgcttcct ttaataaggc catcggtcat 4080 atgcaggaag gttttagaag tacatcttta gcattacaac aaattcaaga tgttgttaat 4140 aagcagagtg ctattcttac tgagactatg gcatcactta ataaaaattt tggtgccatt 4200 tcttctgga ttcaagaaat ctaccagcaa cttgacgcca tacaagcaaa tgctcacgtg 4260 gatcgtctta taactggtag attgtcatca cttctgttt tagcatctgc taagcaggcg 4320 gagtatatta gagtgtcaca acagcgtgag ttagctactc agaagattaa tgagtgtgtt 4380 aagtcacagt ccattaggta ctccttttgt ggtaatggac gacatgtttt aaccataccg 4440 caaaatgcac ctaatggtat agtgtttata cacttttctt acactccaga tagttttgtt 4500 aatgttactg caatagtggg ttttgtgta aagccagcta atgctagtca gtatgcaata 4560 gtacccgcta atggtagggg tatttttata caagttaatg gtagttacta catcactgca 4620 cgagatatgt atatgccaag agctattact gcaggagata tagttacgct tacttcttgt 4680 caagcaaatt atgtaagtgt aaataagacc gtcattacta cattcgtaga caatgatgat 4740 tttgatttta atgacgaatt gtcaaaatgg tggaatgata ctaagcatga gctaccagac 4800 tttgacaaat tcaattacac agtacctata cttgacattg atagtgaaat tgatcgtatt 4860 caaggcgtta tacagggtct taatgactct ctaatagacc ttgaaaaact ttcaatactc 4920 aaaacttata ttaagtggcc ttggtatgtg tggttagcca tagctttgc cactattatc 4980 ttcatcttaa tattaggatg ggttttcttc atgactgggt gttgtggttg ttgttgtgga 5040 tgctttggca tttgcctct aatgagtaag tgtggtaaga aatcttctta ttacacgact 5100 tttgataacg atgtggtaac tgaacaatac agacctaaaa agtctgttta atgatccaaa 5160 gtcccactag tttcttaata gtattaattt tgctttggtg taaacttgta ctaagttgtt 5220 ttagagagtt tattattgcc cttcaacaac taacacaagt tttactccaa attatcgata 5280 gtaatttaca gtctagactg accctttggc acagtctaga ctaatgttaa acttagaagc 5340 aattattgaa accggtgatc aagtgattca aaaaatcagt ttcaatttac agcatatttc 5400 aagtgtatta aacacagaag tatttgaccc ctttgactat tgttattaca gaggaggtaa 5460 tttttgggaa atagagtcag ctgaagattg ttcaggtgat gatgaattta ttgaataagt 5520 cgctagagga gaatggaagt tttctaacgg cactttacat atttgtagga tttttagcat 5580 tttatcttct aggtagagca cttcaagcat ttgtacaggc tgctgatgct tgttgttat 5640 tttggtacac gtggttagta attccaggag ttaagggtac agcctttgta tacaagtata 5700 catatggtag aaaacttaac aattcggaat tagaagcagt tgttgttaac gagtttccta 5760 agaacggttg gaataataaa aatccagcaa atttcaaga tgtccaacga aacaaattgt 5820 actcttgact ttgaacagtc agttgagctt ttagagt ataatttatt tataactgca 5880 ttcttgttgt tctttaccat atacttcag tattggttatg caacgcgtag taagttttatt 5940 tatatactta aaatgagt gttatggtgc ttttggcccc ttaacattgc agtaggtgta 6000 atttcatgta tatacccacc aaacacagga ggtcttgtcg cagcgataat acttactgtg 6060 tttgcgtgtc tttctttgt aggttattgg atccagagta ttagactt taagcggtgt 6120 agatcttggt gttcatttaa cccagaatct aacgccgtag gttcatact cctaactaat 6180 gtcacaat gtattttgc tatagagagt gtgccgatgg tgctttctcc tattataag 6240 aatggtgttc tttattgtga gggtcagtgg cttgctaaat gtgaaccaga ccacttgcct 6300 aaagacatat ttgtatgcac accagataga cgtaatatct atcgtatggt gcagaaatac 6360 actggtgacc aaagcggaaa taagaaaagg ttgctacat ttgtctatgc aaagcagtca 6420 gtagacactg gcagagctaga aagtgtagca acagtggaa gtagccttta cacataaatg 6480 tgtgtgtgta gagagtattt aaaattattc ttcaatagtg cctctatttt aagagcgcgg 6540 aagagtattt gtttgagga tattatata aatcctctt gtttgtact ctctttacaa 6600 gagttattat ttaagcaaca gttttcctt tccttttgttt ggaagaaagt tgttgttaat 6660 ggtgtagaat tccaagtaga aaatggaaaa gtccactacg aaggaaccc cattttcca 6720 aaggttgtt gtaggttgtg gtcccattat aagaaggatt aaatggatta aaccacctac 6780 actacttact tgtaatagg gcgtttggac ttacaagcgc ttaaaata cagacgatga 6840 aatggctgac tagttttgga agagcagtta ttcttgtta taaagcccta ctattaactc 6900 agttaaggt attagatagg ttaatttag atcacggacc aaagcgagtc ttaacgtgtg 6960 gtaggcgagt gcttttatct caattagatt tagtttatag gttggcatat acggcccaccc 7020 aatcgctggt atgaataata gtaagataa tccttttcgc ggagcaatag caaaaagc 7080 gcgaatttat ctgagagaag gattagagtg tgtttactt cttaacaag caggacaagc 7140 agagccttgt cccgcgtgta cctccctagt atttcagggg aaacttgtg aggacacac 7200 agataataat aatctttgt catggcgagc ggtaagacaa ctgggagac agacgcccca 7260 gcgccagtca tcaaactagg agggccaaaa ccacctaaag ttggttcttc tggaaatgct 7320 agctggttc aagcactaaa agccaagaag ttaaattcac ctcctcctaa gtttgaaggt 7380 agcggcgtc ctgataatga aaatcttaaa ttaagccagc aacatgggta ctggagacgt 7440 caagccaggt acaagccagg taaaggcgga agaaaatcag tcccagatgc ttggtacttc 7500 tattacactg gaacaggacc agccgctgac ctgaattggg gtgatagcca agatggtata 7560 gtgtgggttt ctgcaaaggg tgctgatact aaatctagat ctaaccaggg tacaagggat 7620 cctgataagt ttgaccaata cccgctacga ttctcagatg gaggacctga tggtaatttc 7680 cgttgggact tcattccaat aaatcgtggt aggagtggaa gatcaacagc ggcttcatca 7740 gcagcatcta gtagagcacc gtcgcgtgat ggctcgcgtg gacgtagaag cggagctgaa 7800 gatgatctta tagctcgtgc agcaaagatc attcaggatc agcagaagaa gggttctcgc 7860 attactaaag ctaaggccga tgaaatggct catcgccggt attgtaagcg tactatccca 7920 cctggttata aggttgatca agtatttggt ccccgtacta aaggtaagga gggaaatttt 7980 ggtgatgaca agatgaatga ggagggtatt aaggatgggc gcgttacagc aatgctcaac 8040 ctagtcccta gcagccatgc ttgtcttttt ggaagtagag tgacgcccaa acttcaacca 8100 gatgggctgc acttgagatt tgaatttact actgtggttt ctagggatga tccgcagttt 8160 gataattatg tgaaaatttg tgatcagtgt gtcgatggtg tagggactcg gccaaaagac 8220 gatgaaccga gaccaaagtc acgcccaaat tcaagacctg ctacaagaac aagttctcca 8280 gcgccaagac aacagcgtca aaagaaggag aagaagtcaa agaagcagga tgatgaagta 8340 gataaggcat tgacctcaga tgaggagagg aacaatgcac agctggaatt tgatgatgaa 8400 ccgaaagtga ttaactgggg ggattcagca cttggagaga atgagttgta aagctagatt 8460 tccaacttaa catcatggac gtgcgtatgc tgtttttccc tactatagac tttttagcat 8520 attatttttt gctatttgta tggtttatta caggtgaaga ttgtatgtat ttgttgtaca 8580 ctcgtatgtt ctatattg ttttctgtag ttgttattag tgttgttctt gttcttactc 8640 tactgttctc ttttcttat tttagtat caataagaat caaggaagat aggcatgtag 8700 tttgattacc tacatgtcta tcgccaggga aatgtctaat ctgtctactt agtagcctgg 8760 aaacgaacgg tagaccctta gattttaatt tagtttaatt tttagtttag tttaagttag 8820 tttagagtag gtataaagaa gccagtgccg gggccacgcg gagtacgatc gagggtacag 8880 cactaggacg cccactaggg gaagagctaa attttagttt aagttaagtt taattggcta 8940 agtatagtta aaatttatag gctagtatag agttagagca aaaaaaaaaa aaaaaaaaaa 9000 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 9060 aaaaaaaaaa aaaaaaaaag tttaaactta attaagaatt cccttggctc gagttcgaaa 9120 tcggatgccg ggaccgacga gtgcagaggc gtgcaagcga gcttggcgta atcatggtca 9180 tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga 9240 agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg 9300 cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc 9360 caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac 9420 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata 9480 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa 9540 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct 9600 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa 9660 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg 9720 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca 9780 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa 9840 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg 9900 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg 9960 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga 10020 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc 10080 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag 10140 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac 10200 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc 10260 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag 10320 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt 10380 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag 10440 ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca 10500 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact 10560 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca 10620 gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg 10680 tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatcccc 10740 atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg 10800 gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca 10860 tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt 10920 atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc 10980 agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc 11040 ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca 11100 tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa 11160 aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat 11220 tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa 11280 aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtctaagaa 11340 accattatta tcatgacatt aacctataaa aataggcgta tcacgaggcc ctttcgtc 11398 <210> 10 <211> 11398 <212> DNA <213> Artificial sequence <220> <223> Plasmid sequence <400> 10 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 240 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 300 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 360 tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt ggagatcggt acttcgcgaa 420 tgcgtcgaga tcggccgccc gggtaatacg actcactata gggacttaag atagatatta 480 atatatatct attgcactag ccttgcgcta gatttcaaac ttaacaaaac ggacttaaat 540 acctacagct ggtccccata ggtgttccat tgcagtgcac tttagtgccc tggatggcac 600 ctggccacct gtcaggtttt tgttgttaaa atatcattgt tgctggtatc actgcttgtt 660 ttgccgtgtc tcactttata catccgttgc ttgggctacc tagtatccag cgtcctacgg 720 gcgccgtggt cggttcgagt gcgaaggacc tctggttcat ctagcggtag gcgggtgtgt 780 ggaagtagcg cttcagacgt actggttctg ttgcgtgaaa cgcggggtca cctcccccca 840 cataccta agggctttg agcctagcgt tgggctacgt tctcgcacaa ggtcggctat 900 acgacgtttg tagggggtag tgccaaacaa cccctgaggt gacaggttct ggtggtgttt 960 cgaaaacaac aatgtgtgtg ccgcataata tgcgcgttat gcattttgga gcaggtagtg 1020 ataaaggagt ggcgcctggt agtactgttc ttaaacagtg gctccccgaa ggaacactcc 1080 ttgtcgataa tgatattgta gattatgttt ctgacgcaca cgtttctgtg ctttcagatt 1140 gcaataaata taagacagag cacaagtttg atcttgtgat atctgatatg tatacagaca 1200 atgattcaaa aagaaagcat gaaggcgtga tagccaataa tggcaatgat gacgtcttca 1260 tataccttc aagttttcta cgcaataatt tggctctggg aggcagtttt gctgtaaaat 1320 taacagagac aagttggcat gagagtttat atgacattgc acaggattgt gcatggtgga 1380 caatgttttg tacagcagtg aatgcatctt cttcagaagc attcctgatt ggtgttaatt 1440 acttgggtgc aagtgcaaag gttaaagtta gtggaaaaac actgcacgca aattatatat 1500 tttggaggaa ttgtaattat ttacaaacct ctgcttatag tatatttgat gttgctaagt 1560 ttgatttgag attgaaagca acgccagttg ttaatcttaa gactgaacaa aagacagact 1620 tagtctttaa tttgataaag tgtggtaagt tactggtaag agatgttggt aacctctt 1680 ttactagtga ctctttgtg tgcactatgt agtgctgctt tgtatgactc gagttcttac 1740 gtgtactact accaaagtgc cttcagacca cctgatggtt ggcatttaca tgggggtgcg 1800 tatgcggttg ttaatattc tagtgaatct aataatgcag gctcttcatc tgggtgtact 1860 gttggtatta ttcatggtgg tcgtgttgtt aatgcttctt ctatagctat gacggcaccg 1920 tcatcaggta tggcttggtc tagcagtcag ttttgtactg catactgtaa cttttcagat 1980 actacagtgt ttgttacaca ttgtataaa catggtgggt gtcctataac tggcatgctt 2040 caacagcatt ctatacgtgt ttctgctatg aaaaatggcc agcttttcta taatttaaca 2100 gttagtgtag ctaagtaccc tacttttaaa tcatttcagt gtgttaataa tttaacatcc 2160 gtatatttaa atggtgatct tgtttacacc tctaatgaga ccacagatgt tacatctgca 2220 ggtgtttatt ttaaagctgg tggacctata acttataaag ttatgagaga agttagagcc 2280 ctggcttatt ttgttaatgg tactgcacaa gatgttattt tgtgtgatgg gtcacctaga 2340 ggcttgttag catgccagta tatactggc aatttttcag atggctttta tccttttact 2400 aatagtagtt tagttaagca gaagtttatt gtctatcgtg aaaatagtgt tatactact 2460 tttacgttac acaatttcac ttttcataat gagactggcg ccaacccaaa tcctagtggt 2520 gtccagaata ttcaaactta ccaaacacaa acagctcaga gtggttatta taattttaat 2580 ttttccttc tgagtagttt tgtttataag gagtctaatt ttatgtatgg atcttatcac 2640 ccaagttgta atttagact agaaactatt aataatggtt tgtggtttaa ttcactttca 2700 gttagtattg cttacggtcc tcttcaaggt ggttgcaagc aatctgtctt tagtggtaga 2760 gcaacctgtt gttatgctta ctcatatgga ggtcctttgc tgtgtaaagg tgtttattca 2820 ggtgagttag atcataattt tgaatgtgga ctgttagttt atgttactaa gagcggtggc 2880 tctcgtatac aaacagccac tgaaccgcca gttataactc aacacaatta tataatatt 2940 actttaaata cttgtgttga ttataatata tatggcagaa ctggccaggg ttttattact 3000 aatgtaaccg actcagctgt tagttaat tatctagcag acgcaggttt ggctatttta 3060 gatacatctg gttccataga catctttgtc gtacaaagtg aatatggtct taattattat 3120 aaggttaacc cttgcgaaga tgtcaaccag cagtttgtag tttctggtgg taaattagta 3180 ggtattctta cttcacgtaa tgagactggt tcccagcttc ttgagaatca gttttacatc 3240 aaaatcacta atggaacacg tcgttttaga cgttctatta ctgaaagtgt tgaaaattgc 3300 ccttatgtta gttatggtaa gttttgtata aaacctgatg gcagtattgc cacaatagta 3360 ccaaaacagt tagaacagtt tgtggcacct ttacttaatg ttactgaaaa tgtgctcata 3420 cctaacagtt ttaatttaac tgttacagat gagtacatac aaactcggat ggataaggtc 3480 caaattaatt gcctgcagta tatttgtggc aattctctgg agtgcagaaa tttgtttcaa 3540 caatatggtc ctgtttgcga caacatattg tctgtagtaa atagtgttgg tcaaaaagaa 3600 gatatggaac ttttgaattt ctattcttct actaagccgg ctggttttaa tacaccagtt 3660 cttagtaatg ttagcactgg tgagtttaat attactcttt ttttaacaac gcctagtagt 3720 cctagaaggc gttcttttat tgaagacctt ctatttacaa gtgttgaatc tgttggatta 3780 ccaacagatg acgcatacaa aaattgcact gcaggtcctt taggctttct gaaagacctt 3840 gcatgtgctc gtgaatataa tggtttgctt gtgttgcctc ctattataac agcagaaatg 3900 caaactttgt atacaagctc tctagtagct tctatggctt ttggtggtat tactgcagct 3960 ggtgctatac cttttgccac acaactgcag gctagaatta atcacttggg tattacccag 4020 tcacttcttt tgaagaatca agaaaaaatt gctgcttcct ttaataaggc catcggtcat 4080 atgcaggaag gttttagaag tacatcttta gcattacaac aaattcaaga tgttgttaat 4140 aagcagagtg ctattcttac tgagactatg gcatcactta ataaaaattt tggtgccatt 4200 tcttctgtga ttcaagaaat ctaccagcaa cttgacgcca tacaagcaaa tgctcaagtg 4260 gatcgtctta taactggtag attgtcatca ctttctgttt tagcatctgc taagcaggcg 4320 gagtatatta gagtgtcaca acagcgtgag ttagctactc agaagattaa tgagtgtgtt 4380 aagtcacagt ccattaggta ctccttttgt ggtaatggac gacatgtttt aaccataccg 4440 caaaatgcac ctaatggtat agtgtttata cacttttctt acactccaga tagttttgtt 4500 aatgttactg caatagtggg ttttgtgta aagccagcta atgctagtca gtatgcaata 4560 gtacccgcta atggtagggg tatttttata caagttaatg gtagttacta catcactgca 4620 cgagatatgt atatgccaag agctattact gcaggagata tagttacgct tacttcttgt 4680 caagcaaatt atgtaagtgt aaataagacc gtcattacta cattcgtaga caatgatgat 4740 tttgatttta atgacgaatt gtcaaaatgg tggaatgata ctaagcatga gctaccagac 4800 tttgacaaat tcaattacac agtacctata cttgacattg atagtgaaat tgatcgtatt 4860 caaggcgtta tacagggtct taatgactct ctaatagacc ttgaaaaact ttcaatactc 4920 aaaacttata ttaagtggcc ttggtatgtg tggctagcca tagctttgc cactattatc 4980 ttcatcttaa tattaggatg ggttttcttc atgactgggt gttgtggttg ttgttgtgga 5040 tgctttggca tttgcctct aatgagtaag tgtggtaaga aatcttctta ttacacgact 5100 tttgataacg atgtggtaac tgaacaatac agacctaaaa agtctgttta atgatccaaa 5160 gtcccactag tttcttaata gtattaattt tgctttggtg taaacttgta ctaagttgtt 5220 ttagagagtt tattattgcc cttcaacaac taacacaagt tttactccaa attatcgata 5280 gtaatttaca gtctagactg accctttggc acagtctaga ctaatgttaa acttagaagc 5340 aattattgaa accggtgatc aagtgattca aaaaatcagt ttcaatttac agcatatttc 5400 aagtgtatta aacacagaag tatttgaccc ctttgactat tgttattaca gaggaggtaa 5460 tttttgggaa atagagtcag ctgaagattg ttcaggtgat gatgaattta ttgaataagt 5520 cgctagagga gaatggaagt tttctaacgg cactttacat atttgtagga tttttagcat 5580 tttatcttct aggtagagca cttcaagcat ttgtacaggc tgctgatgct tgttgttat 5640 tttggtacac gtggttagta attccaggag ttaagggtac agcctttgta tacaagtata 5700 catatggtag aaaacttaac aattcggaat tagaagcagt tgttgttaac gagtttccta 5760 agaacggttg gaataataaa aatccagcaa atttcaaga tgtccaacga aacaaattgt 5820 actcttgact ttgaacagtc agttgagctt ttagagt ataatttatt tataactgca 5880 ttcttgttgt tctttaccat atacttcag tattggttatg caacgcgtag taagttttatt 5940 tatatactta aaatgagt gttatggtgc ttttggcccc ttaacattgc agtaggtgta 6000 atttcatgta tatacccacc aaacacagga ggtcttgtcg cagcgataat acttactgtg 6060 tttgcgtgtc tttctttgt aggttattgg atccagagta ttagactt taagcggtgt 6120 agatcttggt gttcatttaa cccagaatct aacgccgtag gttcatact cctaactaat 6180 gtcacaat gtattttgc tatagagagt gtgccgatgg tgctttctcc tattataag 6240 aatggtgttc tttattgtga gggtcagtgg cttgctaaat gtgaaccaga ccacttgcct 6300 aaagacatat ttgtatgcac accagataga cgtaatatct atcgtatggt gcagaaatac 6360 actggtgacc aaagcggaaa taagaaaagg ttgctacat ttgtctatgc aaagcagtca 6420 gtagacactg gcagagctaga aagtgtagca acagtggaa gtagccttta cacataaatg 6480 tgtgtgtgta gagagtattt aaaattattc ttcaatagtg cctctatttt aagagcgcgg 6540 aagagtattt gtttgagga tattatata aatcctctt gtttgtact ctctttacaa 6600 gagttattat ttaagcaaca gttttcctt tccttttgttt ggaagaaagt tgttgttaat 6660 ggtgtagaat tccaagtaga aaatggaaaa gtccactacg aaggaaccc cattttcca 6720 aaggttgtt gtaggttgtg gtcccattat aagaaggatt aaatggatta aaccacctac 6780 actacttact tgtaatagg gcgtttggac ttacaagcgc ttaaaata cagacgatga 6840 aatggctgac tagttttgga agagcagtta ttcttgtta taaagcccta ctattaactc 6900 agttaaggt attagatagg ttaatttag atcacggacc aaagcgagtc ttaacgtgtg 6960 gtaggcgagt gcttttatct caattagatt tagtttatag gttggcatat acggcccaccc 7020 aatcgctggt atgaataata gtaagataa tccttttcgc ggagcaatag caaaaagc 7080 gcgaatttat ctgagagaag gattagagtg tgtttactt cttaacaag caggacaagc 7140 agagccttgt cccgcgtgta cctccctagt atttcagggg aaacttgtg aggacacac 7200 agataataat aatctttgt catggcgagc ggtaagacaa ctgggagac agacgcccca 7260 gcgccagtca tcaaactagg agggccaaaa ccacctaaag ttggttcttc tggaaatgct 7320 agctggttc aagcactaaa agccaagaag ttaaattcac ctcctcctaa gtttgaaggt 7380 agcggcgtc ctgataatga aaatcttaaa ttaagccagc aacatgggta ctggagacgt 7440 caagccaggt acaagccagg taaaggcgga agaaaatcag tcccagatgc ttggtacttc 7500 tattacactg gaacaggacc agccgctgac ctgaattggg gtgatagcca agatggtata 7560 gtgtgggttt ctgcaaaggg tgctgatact aaatctagat ctaaccaggg tacaagggat 7620 cctgataagt ttgaccaata cccgctacga ttctcagatg gaggacctga tggtaatttc 7680 cgttgggact tcattccaat aaatcgtggt aggagtggaa gatcaacagc ggcttcatca 7740 gcagcatcta gtagagcacc gtcgcgtgat ggctcgcgtg gacgtagaag cggagctgaa 7800 gatgatctta tagctcgtgc agcaaagatc attcaggatc agcagaagaa gggttctcgc 7860 attactaaag ctaaggccga tgaaatggct catcgccggt attgtaagcg tactatccca 7920 cctggttata aggttgatca agtatttggt ccccgtacta aaggtaagga gggaaatttt 7980 ggtgatgaca agatgaatga ggagggtatt aaggatgggc gcgttacagc aatgctcaac 8040 ctagtcccta gcagccatgc ttgtcttttt ggaagtagag tgacgcccaa acttcaacca 8100 gatgggctgc acttgagatt tgaatttact actgtggttt ctagggatga tccgcagttt 8160 gataattatg tgaaaatttg tgatcagtgt gtcgatggtg tagggactcg gccaaaagac 8220 gatgaaccga gaccaaagtc acgcccaaat tcaagacctg ctacaagaac aagttctcca 8280 gcgccaagac aacagcgtca aaagaaggag aagaagtcaa agaagcagga tgatgaagta 8340 gataaggcat tgacctcaga tgaggagagg aacaatgcac agctggaatt tgatgatgaa 8400 ccgaaagtga ttaactgggg ggattcagca cttggagaga atgagttgta aagctagatt 8460 tccaacttaa catcatggac gtgcgtatgc tgtttttccc tactatagac tttttagcat 8520 attatttttt gctatttgta tggtttatta caggtgaaga ttgtatgtat ttgttgtaca 8580 ctcgtatgtt ctatattg ttttctgtag ttgttattag tgttgttctt gttcttactc 8640 tactgttctc ttttcttat tttagtat caataagaat caaggaagat aggcatgtag 8700 tttgattacc tacatgtcta tcgccaggga aatgtctaat ctgtctactt agtagcctgg 8760 aaacgaacgg tagaccctta gattttaatt tagtttaatt tttagtttag tttaagttag 8820 tttagagtag gtataaagaa gccagtgccg gggccacgcg gagtacgatc gagggtacag 8880 cactaggacg cccactaggg gaagagctaa attttagttt aagttaagtt taattggcta 8940 agtatagtta aaatttatag gctagtatag agttagagca aaaaaaaaaa aaaaaaaaaa 9000 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 9060 aaaaaaaaaa aaaaaaaaag tttaaactta attaagaatt cccttggctc gagttcgaaa 9120 tcggatgccg ggaccgacga gtgcagaggc gtgcaagcga gcttggcgta atcatggtca 9180 tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga 9240 agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg 9300 cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc 9360 caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac 9420 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata 9480 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa 9540 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct 9600 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa 9660 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg 9720 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca 9780 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa 9840 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg 9900 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg 9960 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga 10020 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc 10080 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag 10140 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac 10200 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc 10260 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag 10320 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt 10380 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag 10440 ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca 10500 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact 10560 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca 10620 gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg 10680 tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatcccc 10740 atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg 10800 gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca 10860 tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt 10920 tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt 10920 atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc 10980 atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc 10980 agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc 11040 agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc 11040 ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca 11100 ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca 11100 tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa 11160 tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa 11160 aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat 11220 aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat 11220 tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa 11280 tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa 11280 aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtctaagaa 11340 aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtctaagaa 11340 accattatta tcatgacatt aacctataaa aataggcgta tcacgaggcc ctttcgtc 11398 accattatta tcatgacatt aacctataaa aataggcgta tcacgaggcc ctttcgtc 11398 <210> 11 <211> 1155 <212> PRT <213> Infectious bronchitis virus of poultry <400> 11 Met Leu Asp Lys Pro Leu Leu Leu Val Thr Leu Trp Tyr Ala Leu Cys Met Leu Asp Lys Pro Leu Leu Leu Val Thr Leu Trp Tyr Ala Leu Cys 1 5 10 15 Ser Ala Leu Leu Tyr Asp Asn Asn Thr Tyr Val Tyr Tyr Tyr Gln Ser 20 25 30 Ala Phe Arg Pro Gly Pro Gly Trp His Leu Tyr Gly Gly Ala Tyr Ala 35 40 45 Val Asp Arg Val Phe Asn Glu Thr Asn Asn Ala Gly Ser Ala Ser Asp 50 55 60 Cys Thr Ala Gly Thr Phe Tyr Glu Ser His Asn Ile Ser Ala Ser Ser 65 70 75 80 Val Ala Met Thr Val Pro His Asn Gly Met Ser Trp Ser Ala Ser Gln 85 90 95 Phe Cys Thr Ala His Cys Asn Phe Ser Asp Phe Thr Val Phe Val Thr 100 105 110 His Cys Phe Lys Asn Gln Leu Gly Ser Cys Pro Leu Thr Gly Met Ile 115 120 125 Pro Gln Asn His Ile Arg Ile Ser Ala Met Arg Asp Gly Val Leu Phe 130 135 140 Tyr Asn Leu Thr Val Ser Val Ser Lys Tyr Pro Arg Phe Lys Ser Leu 145 150 155 160 Gln Cys Val Ser Asn Ser Thr Ser Val Tyr Val Asn Gly Asp Leu Val 165 170 175 Phe Thr Ser Asn Glu Thr Ser Tyr Val Thr Gly Ala Gly Val Tyr Phe 180 185 190 Lys Ser Gly Gly Pro Val Thr Tyr Lys Val Met Lys Glu Val Lys Ala 195 200 205 Leu Ala Tyr Phe Ile Asn Gly Thr Ala Gln Glu Val Ile Leu Cys Asp 210 215 220 Asn Ser Pro Arg Gly Leu Leu Ala Cys Gln Tyr Asn Thr Gly Asn Phe 225 230 235 240 Ser Asp Gly Phe Tyr Pro Phe Thr Asn His Ser Leu Val Lys Asp Arg 245 250 255 Phe Ile Val Tyr Arg Glu Ser Ser Thr Asn Thr Thr Leu Lys Leu Thr 260 265 270 Asn Phe Ser Phe Thr Asn Val Ser Asn Ala Ser Pro Asn Ser Gly Gly 275 280 285 Val Asp Thr Phe Gln Leu Tyr Gln Thr Ser Thr Ala Gln Asp Gly Tyr 290 295 300 Tyr Asn Phe Asn Leu Ser Phe Leu Ser Ser Phe Val Tyr Lys Pro Ser 305 310 315 320 Asp Phe Met Tyr Gly Ser Tyr His Pro His Cys Lys Phe Arg Pro Glu 325 330 335 Asn Ile Asn Asn Gly Leu Trp Phe Asn Ser Leu Ser Val Ser Leu Thr 340 345 350 Tyr Gly Pro Ile Gln Gly Gly Cys Lys Gln Ser Val Phe Ser Asn Arg 355 360 365 Ala Thr Cys Cys Tyr Ala Tyr Ser Tyr Gln Gly Pro Ser Arg Cys Lys 370 375 380 Gly Val Tyr Arg Gly Glu Leu Thr Gln Tyr Phe Glu Cys Gly Leu Leu 385 390 395 400 Val Tyr Val Thr Lys Ser Asp Gly Ser Arg Ile Gln Thr Arg Ser Glu 405 410 415 Pro Leu Val Leu Thr Gln Tyr Asn Tyr Asn Asn Ile Thr Leu Asn Lys 420 425 430 Cys Val Glu Tyr Asn Ile Tyr Gly Arg Val Gly Gln Gly Phe Ile Thr 435 440 445 Asn Val Thr Glu Ala Thr Ala Asn Tyr Ser Tyr Leu Ala Asp Gly Gly 450 455 460 Leu Ala Ile Leu Asp Thr Ser Gly Ala Ile Asp Ile Phe Val Val Gln 465 470 475 480 Gly Ala Tyr Gly Leu Asn Tyr Tyr Lys Val Asn Pro Cys Glu Asp Val 485 490 495 Asn Gln Gln Phe Val Val Ser Gly Gly Asn Leu Val Gly Ile Leu Thr 500 505 510 Ser His Asn Glu Thr Gly Ser Glu Ser Ile Glu Asn Gln Phe Tyr Ile 515 520 525 Lys Leu Thr Asn Gly Thr Arg Arg Ser Arg Arg Ser Val Thr Gly Asn 530 535 540 Val Thr Asn Cys Pro Tyr Val Ser Tyr Gly Lys Phe Cys Ile Lys Pro 545 550 555 560 Asp Gly Ser Leu Ser Ile Ile Val Pro Gln Glu Leu Glu Gln Phe Val 565 570 575 Ala Pro Leu Phe Asn Val Thr Glu His Val Leu Ile Pro Asp Ser Phe 580 585 590 Asn Leu Thr Val Thr Asp Glu Tyr Ile Gln Thr Arg Met Asp Lys Val 595 600 605 Gln Ile Ile Cys Leu Gln Tyr Val Cys Gly Asn Ser Ile Glu Cys Arg 610 615 620 Lys Leu Phe Gln Gln Tyr Gly Pro Val Cys Asp Asn Ile Leu Ser Val 625 630 635 640 Val Asn Gly Val Gly Gln Arg Glu Asp Met Glu Leu Leu Ser Phe Tyr 645 650 655 Ser Ser Thr Lys Pro Ser Gly Tyr Asn Thr Pro Ile Phe Asn Asn Val 660 665 670 Ser Thr Gly Asp Phe Asn Ile Ser Leu Leu Leu Thr Pro Pro Asn Ser 675 680 685 Pro Thr Gly Arg Ser Phe Ile Glu Asp Leu Leu Phe Thr Ser Val Glu 690 695 700 Ser Val Gly Leu Pro Thr Asp Glu Glu Tyr Lys Lys Cys Thr Ala Gly 705 710 715 720 Pro Leu Gly Phe Val Lys Asp Leu Val Cys Ala Arg Glu Tyr Asn Gly 725 730 735 Leu Leu Val Leu Pro Pro Ile Ile Thr Ala Glu Met Gln Thr Met Tyr 740 745 750 Thr Ser Ser Leu Val Ala Ser Met Ala Leu Gly Gly Ile Thr Ala Ala 755 760 765 Gly Ala Ile Pro Phe Ala Thr Gln Leu Gln Ala Arg Ile Asn His Leu 770 775 780 Gly Ile Thr Asn Ser Leu Leu Leu Lys Asn Gln Glu Lys Ile Ala Ala 785 790 795 800 Ser Phe Asn Lys Ala Ile Gly His Met Gln Glu Gly Phe Lys Ser Thr 805 810 815 Ser Leu Ala Leu Gln Gln Ile Gln Asp Val Val Asn Lys Gln Ser Ser 820 825 830 Ile Leu Thr Glu Thr Met Gln Ser Leu Asn Lys Asn Phe Gly Ala Ile 835 840 845 Ser Ser Val Ile Gln Asp Ile Tyr Gln Gln Leu Asp Ala Ile Gln Ala 850 855 860 Asp Ala Gln Val Asp Arg Leu Ile Thr Gly Arg Leu Ser Ser Leu Ser 865 870 875 880 Val Leu Ala Ser Ala Lys Gln Ala Glu Tyr His Arg Val Ser Gln Gln 885 890 895 Arg Glu Leu Ala Thr Gln Lys Ile Asn Glu Cys Val Lys Ser Gln Ser 900 905 910 Asn Arg Tyr Ser Phe Cys Gly Asn Gly Arg His Val Leu Thr Ile Pro 915 920 925 Gln Asn Ala Pro Asn Gly Ile Val Phe Ile His Phe Thr Tyr Thr Pro 930 935 940 Glu Ser Phe Val Asn Val Thr Ala Ile Val Gly Phe Cys Val Asn Pro 945 950 955 960 Ala Asn Ala Ser His Tyr Ala Ile Val Pro Val Asn Gly Arg Gly Val 965 970 975 Phe Ile Glu Val Asn Gly Ser Tyr Tyr Ile Thr Ala Arg Asp Met Tyr 980 985 990 Met Pro Arg Asp Ile Thr Ala Gly Asp Ile Val Thr Leu Thr Ser Cys 995 1000 1005 Gln Ala Asn Tyr Val Asn Val Asn Lys Thr Val Ile Asn Thr Phe 1010 1015 1020 Val Glu Asp Asp Asp Phe Asp Phe Tyr Asp Glu Leu Ser Lys Trp 1025 1030 1035 Trp Asn Asp Thr Lys His Glu Leu Pro Asp Phe Asp Glu Phe Asn 1040 1045 1050 Tyr Thr Val Pro Val Leu Asn Ile Ser Asn Glu Ile Asp Arg Ile 1055 1060 1065 Gln Gln Val Ile Gln Gly Leu Asn Asp Ser Leu Ile Asp Leu Glu 1070 1075 1080 Thr Leu Ser Ile Leu Lys Thr Tyr Ile Lys Trp Pro Trp Tyr Val 1085 1090 1095 Trp Leu Ala Ile Ala Phe Leu Thr Ile Ile Phe Ile Leu Val Leu 1100 1105 1110 Cys Trp Ile Phe Phe Met Thr Gly Cys Cys Gly Cys Cys Cys Gly 1115 1120 1125 Cys Phe Gly Ile Ile Pro Leu Met Ser Lys Cys Gly Lys Lys Ser 1130 1135 1140 Ser Tyr Tyr Thr Thr Phe Asp Asn Asp Val Val Thr 1145 1150 1155 <210> 12 <211> 11860 <212> DNA <213> Artificial sequence <220> <223> Plasmid sequence <400> 12 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 240 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 300 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 360 tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt ggagatcggt acttcgcgaa 420 tgcgtcgaga tgagctctaa tacgactcac tatagggact taagtgtgat ataaatatat 480 atcatacata ctagccttgt gctagatttc caacttaaca aaacggactt aaatacctac 540 agttggtccc tataggtgtt ccattgcagt gcactttagt gccctggatg gcacctggcc 600 acctgtcagg tttttgttat taaataata ttgttgctgg tatcactgct tgttttgccg 660 tgtctcactt tatacatccg ttgcttgggc tacctagtat ccagcgtcct actggcgttg 720 tggtcggttc gagtgcgaag aacctctggt tcatctagcg gtacgcgggt gtgtggaagt 780 agcgcttcag acgtaccggt tctgttgcgt gaaatacggg gtcacctccc cccacatacc 840 tctaagggct tttgagccta gcgttgggct acgttctcgc acaaggtcgg ctaacggcg 900 tttgtagggg gtagtgccaa acaacccctg aggtgacagg ttctggtggt gtttcgaaaa 960 caacaatgtg tgtgccgcat aatatgcgag taatgcattt tggagcagga agtgacaaag 1020 gagtggcccc aggtagcgct gttcttaggc agtggcttcc cgaaggtaca ctccttgtcg 1080 ataatgatat tgtagattat gtatctgatg cacatgtctc tgtgctttca gattgcaata 1140 aatgtaaaac agagcacaag tttgatcttg tgatatctga tatgtataca gataatgatt 1200 caaagagaaa gcatgaaggc gttgtagcca ataacggcaa tgatgacgtc ttcatatacc 1260 tttcaaactt tcttcgtaac aatttagctc tgggaggcag ttttgccgta aaagtaacag 1320 agacaagttg gcatgagagt ttatatgaca ttgcacagga ttgtgcatgg tggacaatgt 1380 tttgtacagc agtgaatgct tcttcgtcag aagcattctt gattggtgtt aattatttgg 1440 gtgcaagtga aaaggttaga gttagtggta aaaccctgca cgcaaattat atattttgga 1500 ggaattgtaa ttattacaa acttcagcct atagtatatt tgacgttgct aagttttgatt 1560 tgaaattaaa agcaacgcca gttgtgaatt tgaaaactga acaaaagaca gacttagtct 1620 ttaatttaat taagtgtggt aagttactgg taagagatgt tggacaaacc gcttttacta 1680 gtgactcttt ggtatgcact atgtagtgct ttgctctatg ataataatta tattggtgat 1740 tttagatgta tccagcttgt gaactcaaac ggtgctaatg ttagtgctcc aagcattagc 1800 actgagaccg ttgaagtttc acaaggcctg gggacatatt atgtgttaga tcgagtttat 1860 ttaaatgcca cattattgct tactggttac tacccggtcg atggttctaa gtttagaaac 1920 ctcgctctta cgggaactaa ctcagttagc ttgtcgtggt ttcaaccacc ctatttaagt 1980 cagtttaatg atggcatatt tgcgaaggtg cagaacctta agacaagtac gccatcaggt 2040 gcaactgcat attttcctac tatagttata ggtagtttgt ttggctatac ttcctatacc 2100 gttgtaatag agccatataa tggtgttata atggcctcag tgtgccagta taccatttgt 2160 cagttacctt acactgattg taagcctaac actaatggta ataagcttat agggttttgg 2220 cacacggatg taaaaccccc aatttgtgtg ttaaagcgaa atttcacgct taatgttaat 2280 gctgatgcat tttattttca tttttaccaa catggtggta ctttttatgc gtactatgcg 2340 gataaaccct ccgctactac gtttttgttt agtgtatata ttggcgatat tttaacacag 2400 tattatgtgt tacctttcat ctgcaaccca acagctggta gcacttttgc tccgcgctat 2460 tgggttacac ctttggttaa gcgccaatat ttgtttaatt tcaaccagaa gggtgtcatt 2520 actagtgctg ttgattgtgc tagtagttat accagtgaaa taaaatgtaa gacccagagc atgttaccta gcactggtgt ctatgagtta tccggttata cggtccaacc agttggagtt 2640. gtataccggc gtgttgctaa cctcccagct tgtaatatag aggagtggct tactgctagg tcagtcccct cccctctcaa ctggggagcgt aagacttttc agaattgtaa ttttaattta agcagcctgt tacgttatgt tcaggctgag agtttgtttt gtaatatat cgatgcttcc aaagtgtatg gcaggtgctt tggtagtatt tcagttgata agtttgctgt accccgaagt 2880 aggcaagttg atttacagct tggtaactct ggatttctgc agactgctaa ttataagatt 3000. 3000. 3000. 3000. 3000. 3000. 3000. 3000. 3000. 3000 aacaaccata acccctcgtc ttggaatagg aggtatggct ttaatgatgc tggcgtcttt ggcaaaaacc aacatgacgt tgtttacgct cagcaatgtt ttactgtaag atctagttat tgcccgtgtg ctcaaccgga catagttagc ccttgcacta ctcagactaa gcctaagtct 3180. gcttttgtta atgtgggtga ccattgtgaa ggcttaggtg ttttagaaga taattgtggc aatgctgatc cacataaggg ttgtatctgt gccaacaatt catttattgg atggtcacat 3300 gatacctgcc ttgttaatga tcgctgccaa atttttgcta atatattgtt aaatggcatt 3360 aatagtggta ccacatgttc cacagatttg cagttgccta atactgaagt ggttactggc 3420 atttgtgtca aatatgacct ctacggtatt actggacaag gtgtttttaa agaggttaag 3480 gctgactatt ataatagctg gcaaaccctt ctgtatgatg ttaatggtaa tttgaatggt 3540 tttcgtgatc ttaccactaa caagacttat acgataagga gctgttatag tggccgtgtt 3600 tctgctgcat ttcataaaga tgcacccgaa ccggctctgc tctatcgtaa tataaattgt 3660 agctatgttt ttagcaataa tatttcccgt gaggagaacc cacttaatta ctttgatagt 3720 tatttgggt gtgttgttaa tgctgataac cgcacggatg aggcgcttcc taattgtgat 3780 ctccgtatgg gtgctggctt atgcgttgat tattcaaaat cacgcagggc tcaccgatca 3840 gttctactg gctatcggtt aactacattt gagccataca ctccgatgtt agttaatgat 3900 agtgtccaat ccgttgatgg attatatgag atgcaaatac caaccaattt tactattggg 3960 caccatgagg agttcattca aactagatct ccaaaggtga ctatagattg tgctgcattt 4020 gtctgtggtg ataacactgc atgcaggcag cagttggttg agtatggctc tttctgtgtt 4080 aatgttaatg ccattcttaa tgaggttaat aacctcttgg ataatatgca actacaagtt 4140 gctagtgcat taatgcaggg tgttactata agttcgagac tgccagacgg catctcaggc 4200 cctatagatg acattaattt tagtcctcta cttggatgca taggttcaac atgtgctgaa 4260 gacggcaatg gacctagtgc aatccgaggg cgttctgcta tagaggatttt gttatttgac 4320 aaggtcaaat tatctgatgt tggctttgtc gaggcttata ataattgcac cggtggtcaa 4380 gaagttcgtg acctcctttg tgtacaatct tttaatggca tcaaagtatt acctcctgtg 4440 ttgtcagaga gtcagatctc tgctacaca accggtgcta ctgcggcagc tatgttccca 4500 ccgtggtcag cagctgccgg tgtgccattt agtttaagtg ttcaatatag aattaatggt 4560 ttaggtgtca ctatgaatgt gcttagtgag aaccaaaaga tgattgctag tgcttttaac 4620 aatgcgctgg gtgctatcca ggatgggttt gatgcaacca attctgcttt aggtaagatc 4680 cagtccgttg ttaatgcaa tgctgaagca ctcaataact tactaatca actttctaac 4740 aggtttggtg ctattagtgc ttctttacaa gaattctaa ctcggcttga ggctgtagaa 4800 gcaaaagcccc agatagatcg tcttattaat ggcaggttaa ctgcacttaa tgcgtatata 4860 tccaagcaac ttagtgatag tacgcttatt aaagttagtg ctgctcaggc catagaaag 4920 gtcaatgagt gcgttaagg ccaaaccacg cgtattaatt tctgtggcaa tgtaatcat 4980 atattatctc ttgtccagaa tgcgcttat ggcttatatt ttacactt cagctatgtg 5040 ccaatatcct ttacaccgc aaatgtgagt cctggactt gcattctctg tgatagagga 5100 ttagcaccta aagctggata tttgttca gatgatggag aatggagtt cacaggcagt 5160 tcatattact accctgaacc cattacagat aaaaacagtg tcattatgg tagttgcgca 5220 gtaaactaca caaggcacc tgaagttttc ttgaacactt caataccta tccacccgac 5280 tttaggagg agttagataa atggttttaag atcagacgt ctattgcgcc tgatttatct 5340 ctcgatttcg agaagttaaa tgttacttg ctggacctga cgtatgagat gaacaggatt 5400 caggatgcaa ttaagaagtt aaatgagagc tacatcaacc tcaaggaagt tggcacatat 5460 gaaatgtatg tgaaatggcc ttggtatgtg tggcttgcca ttgcattcct taccattatt 5520 tttattctgg tactttgttg gatatttttc atgaccggtt gttgcggttg ttgttgtgga 5580 tgctttggta tcataccgtt aatgagtaag tgtggtaaga aatcttctta ctacacgact 5640 tttgataatg atgtggtaac ttaacaatac agacctaaaa agtctgttta atgattaaaa 5700 gtcccacatc ttttctaata ttattaattc ttctttggtg taaacttgca ttaagttgtt 5760 ttaaagagtg tgttataaca ctccagcaac tagtacaaat tttactccaa attattaata 5820 gtaacttaca atctagactt ctgctttggc acagtctaga ctaatgttag attttgaagc 5880 aattattgaa actggtcagc aaataactca acaaattagt ttctatttac agcatatttc 5940 aagggtgcta agtactgaat tatttgaccc ctttgaagtt tgtgtttaca gaggaggtaa 6000 ttgttgggag ttagagtcag ctgacgagtt ttcaggtgat gacgaatata ttgagtagat 6060 cgctcgagga gaacggaagt tttctaacag cggtttacgt gtttttagga tttttagcac 6120 tttatctact aggtagcg cttcaagctt ttgtacaagc ggctgacgct tgttgtcttt 6180 tttggtatac atgggtagta gttcctggag ccaagggcac agcctttgtt fathercata catatggtaa aaaacttaac aaaccggagt tagaaacggt tattgttaac gaatttccaa aaaacggttg gaatatgga fatherccatc aattgtactc ttggtactga acaagcagtt cagcttttta aggate tctgtttgta actgcattcc tgttgttttt aaccatacta cttcagtatg gatacgcaac taggcaag gttatttaca tactgaaaat gatagtgtta tggtgctttt ggccccttaa cattgcagta ggtgtaatct catgtatata cccaccaaac acaggaggtc ttgtcgcagc gataattctt acagtgtttc cgtgtctttc ttttataggt 6600. tattggatcc agagtattag actttttaag cggtgcaggt catggtggtc atttaacccc gaatctatg ccgtaggttc aatactccta actatggtc aacaatgtaa ttttgctata gagagtgtgc cgatggtgct ttctcctatt father gtgctcttta ttgcgagggt 6780 cagtggcttg ctaaatgtga accagaccac ttgcctagg fatheratttgt atgcacaccg gatgacgta atatctatcg tattggtgca aaatactg gtgaccaag cggagtag 6900 aaaaggtttg ccacattgt ctatgcaaag cagtcagtag atactggcga gctagaaagt 6960 gtgtcagcag taggagg tctttacaca taaatgtgtg tgtgtagaga gtatttaaaa 7020 ttattctttg acagtgccctc cgttttaaga gcgcggaaga gtattattt tgaggatatt 7080 atataaatc ctctttgttt catactctcc ttcaggagt tattatta aaaacagttt 7140 ttccactctt tgtgccaaa aacaattgtt gttaatggtg taaccttca ggtagacaat 7200 ggaaaagtct actacgaagg aagaccaatt ttccaaaag gttgttgtag ttgtggtcc 7260 aattataaga agattagaa taatttaacc acctacaca cttatttta caatggcgt 7320 tttaggttac aaacgcttaa siaatacgga tgatgaatg gctgactagt ttggaagg 7380 ctttcatctc ctgttataa tccctattac taactcatt aagagtatta gataggttaa 7440 ttttagatca cggaccaag cgcacattaa cgtgtgctag gcgagtgctt ttagttcaat 7500 tagattagt ttataggttg gcttatacgc ccacccaatc gctggtatga atatagtaa 7560 agataatcct tttcgcggag caatagcaag aaaagcgcga atttatctga gaggaggatt 7620 agattgtgtt tactttctta acaaagcagg acaagcagag ccttgtcccg cgtgtacctc 7680 cctagtattc caagggaaaa cttgtgagga acactattat aataacaatc ttttgtcatg 7740 gcaagcggta aggcaactgg aaagacagac gccccagcgc cagtcatcaa actaggagga 7800 ccaaagccac ctaaagttgg ttcttctgga aatgcatcat ggtttcaacc gataaaggcc 7860 aagaagctaa attcacctgt gcctaaattt gacggtagtg gtgttcctga aaatgaaaat 7920 ctcaagtcaa gccagcaaca tggatactgg agacgccaac acaggtttaa gcctggcaaa 7980 ggtggaagaa aaccagtccc tgatgcttgg tacttttact acactggaac aggaccggcc 8040 gccgacctga attggggtga aactcaagat ggtatagtgt gggttgctgc aaagggtgct 8100 gatactaaat ctagatcaaa ccagggtaca agggatcctg ataagtttga ccaataccca 8160 ctacgattct cagatggagg accggatggt aatttccgtt gggacttcat accaataaat 8220 cgtggtagga gtgggagatc aacagcagct tcatcagcag catctagtag agcaccatct 8280 8340 aagattatac aggaccagca aaagaagggt gcgcgcatta ccaaggctaa ggctgatgaa 8400 8460 tttggtcccc gtactaaagg taggaaagga aattttggtg atgacaagat gaatgaggaa 8520 ggtgttaagg atgggcgtgt tacggcaatg ctcaacctag tccctagcag tcatgcttgt 8580 ctttttggaa gtagggtgac gcccaaactg cagccagatg gttttcacct gagatttgaa 8640 tttactactg tggtgtcacg tgatgatccg cagtttgata attatgtgaa aatttgtgat 8700 cagtgtgtcg atggtgtagg gacgcgtcca areacgatg aatcgagacc aaagtcacgc 8760 ccaaattcaa gacctgcaac tagaggaaat tctccagcgc cgagacaaca gcgcccaaag 8820 aaggagaaaa agcccaagaa gcaggatgat gaagtagata aggcattgac ctcagatgag 8880 gaggaaca atgcacagct ggaatttgat gatgaaccca aggtgattaa ctggggggac 8940 tctgcactag gtgaaatga actttgatta acataatgga cttgctgcat ttgctgtcac 9000 atttgttaa atattatttt tgtgttttac tatcaattat tacaggtatt gattgtgatt 9060 atgttcaata cttaagcttc ttctggttgc ttttgcttg ttgtattgtt gctgtgcttt 9120 ttattattgt gattctcatt agtttgcttt atcgtagaaa ttcaatagta agagttaagg 9180 aagataggca tgtagcttag cacctacatg tctatcgcca gggaaatgtc taatctgtct 9240 acttagtagc ctggaaacga acggtagacc cttagatttt aatttagttt aatttttagt 9300 ttagtttaag ttagtttaga gtaggtataa agatgccagt gccggggcca cgcgtagtac 9360 gaccgagggt acagcactag gacgcccact aggggaagag ctaaatttta gtttaagtta 9420 agtttaattg gctaaatata gttaaaattt ataggctagt atagagttag agcaaaaaaa 9480 aaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa 9540 aaaaaaaaaa aaaaaaaaa aaaaaaaaa aaagcggccg catcggatgc cgggaccgac 9600 gagtgcagag gcgtgcaagc gagcttggcg taatcatggt catagctgtt tcctgtgtga 9660 aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc 9720 tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc 9780 cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc 9840 ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt 9900 cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca 9960 ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa 10020 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat 10080 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc 10140 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc 10200 gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt 10260 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac 10320 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg 10380 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca 10440 gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc 10500 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa 10560 accaccgctg gtagcggtgg ttttttgtt tgcaagcagc agattacgcg cagaaaaaaa 10620 ggatctcaag aagatccttt gatctttct acggggtctg acgctcagtg gaacgaaaac 10680 tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta 10740 attaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt 10800 taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata 10860 gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc 10920 agtgctgcaa tgataccgc agacccacgc tcaccggctc cagatttatc agcaataaac 10980 cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag 11040 tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac 11100 gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc 11160 agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg 11220 gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc 11280 atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct 11340 gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc 11400 tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc 11460 atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc 11520 agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc 11580 gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca 11640 cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt 11700 tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt 11760 ccgcgcacat ttccccgaaa agtgccacct gacgtctaag aaaccattat tatcatgaca 11820 ttaacctata aaaataggcg tatcacgagg ccctttcgtc 11860 <210> 13 <211> 6905 <212> DNA <213> Artificial Sequence <220> <223> Plasmid Sequence <400> 13 ttaattaagt gtggtaagtt actggtaaga gatgttggac aaaccgcttt tactagtgac 60 tctttggtat gcactatgta gtgctttgct ctatgataat aatacttacg tttactacta 120 ccaaagtgcc tttaggcctg gtccaggttg gcacctatat gggggtgctt atgcagtaga 180 tagggttttt aatgaaacca acaatgcagg cagtgcatct gattgcactg ctggtacttt 240 ttatgaaagc cataatattt ctgcttcttc tgtagccatg acagtaccac ataatggtat 300 gtcttggtca gcttcacaat tttgtacagc tcattgtaac ttctcagact ttacagtgtt 360 cgttacgcat tgttttaaaa atcaactcgg tagttgtccc ttgacaggta tgattcctca 420 gaatcatatt cgtatttctg ctatgagaga tggagttttg ttttataact taacagttag 480 cgtatctaaa taccctagat ttaaatcgct tcaatgtgtt agcaattcta catctgtcta 540 tgtaaatggt gaccttgttt tcacttctaa tgaaacttct tacgttacgg gtgcaggcgt 600 ttattttaaa agtggtgggc ctgtaactta taaagttatg aaagaagtta aagccctagc 660 ctactttatt aatggtaccg cacaagaggt tattttatgt gataactcac ctagaggtttt 720 gcttgcatgt cagtataaca ctggtaattt ttcagatgga ttctaccctt ttactaatca 780 ttctttagtt aaggataggt ttattgtata tcgagaaagt agcactaaca ctactttaaa 840 gttaactaat ttcagtttta ctaatgtaag taatgcttct cctaattcag gtggcgttga 900 tactttccaa ttatatcaaa caagtactgc tcaggatggt tattataatt ttaatttatc 960 atttctgagt agttttgtgt ataaaccatc tgattttatg tatgggtcat accacccaca 1020 ttgtaagttt agaccagaga atattaataa tggcttatgg tttaattcat tatctgtgtc 1080 acttacttac ggacccattc aaggtggttg taagcaatct gtttttagta atagagcaac 1140 ttgttgctat gcttattctt atcaagggcc tagtagatgt aagggtgttt atagagggga 1200 gctaacgcaa tactttgaat gtggacttct agtttacgta actaagagtg atggctctcg 1260 tatacaaact agaagtgaac cactggtgtt aactcaatat aattataaca acattacttt 1320 aaataagtgt gttgagtata atatatatgg tagggttggt caaggtttta ttactaatgt 1380 aactgaagca actgctaatt atagttatct agcagatggt ggtttagcta ttttagatac 1440 ctcaggagcc atagacatat ttgttgttca aggtgcatat ggtcttaatt attataaggt 1500 taatccctgt gaagatgtta accaacagtt tgtagtgtct ggtggcaact tagttggcat 1560 tcttacatct cataatgaaa caggttctga atctattgag aaccagtttt acatcaaact 1620 cactaacgga acacgtcgct ctagacgttc tgttactggg aatgttacaa attgccctta 1680 tgttagttat ggcaagtttt gtataaaacc agatggttct ttatctataa tagtaccaca 1740 agaattagaa cagtttgtgg cgcctttatt caatgttact gagcatgtgc tcatacctga 1800 tagttttaat ttaactgtca cagatgagta catacaaact cgtatggata aggttcaaat 1860 tatttgcctt cagtatgttt gtggtaattc tattgaatgc agaaagttgt ttcagcagta 1920 tggacctgtt tgtgataata tattgtctgt tgtaaatggt gtaggtcaaa gagaggatat 1980 ggaactttta agtttctatt cttctactaa acctagtggt tacaatacac caatttttaa 2040 taatgttagc actgtgact ttatatttc gctcctacta acaccaccta atagtcctac 2100 tgggcgctct tttattgaag atctctctt tacaagtgta gatctgttg gattaccac 2160 tgatgaagg tataaaaagt gtacagcagg acctttaggt ttgttaag accttgttg 2220 tgctagagag tataatggtt tgctcgttct gcctcctatt attactgcgg aaatgcaaac 2280 catgtatact agttctttag tagctctat ggctttaggt gcattactg cagctggtgc 2340 tatacctttt gctacacaac tgcaggccag aattaccat ttggtatta ctaatctct 2400 tttgttgaaa aaccaagaaaaattgctgc ttccttaat aaggccatcg gtcatatgca 2460 ggaagggttt aaagtactt cttagcatt acacatt caatgttg ttaaaca 2520 gagttctatt cttacagaga ctagcaatc acttataa aattttggtg ctatttccctc 2580 tgtaattca gatattacc agcaactaga tgctattcag gcagatgctc agggttgatcg 2640 tcttattaca ggtagactct cttcactac tgtttagct tctgctaac aggcagagta 2700 tcatagagtg tcacaacagc gtgagttggc cactcagaaa attaatgagt gtgttaagtc 2760 tcagtctaat aggtattcat tttgtggtaa tggtagacat gttctaacca taccacagaa 2820 tgcacccaat ggcatagtgt ttatacactt tacatacact ccagagagtt ttgttaatgt 2880 tacggcaata gtagggtttt gcgtaaaccc agctaatgct agtcattatg caatagtgcc 2940 tgttaatggc aggggtgttt ttatagaagt taatggtagt tactatatca ctgctcgtga 3000 tatgtatatg ccaagagata ttactgcagg agacatagtc actttgactt cttgtcaagc 3060 aaactatgtt aatgtaaata aaaccgtcat taacactttt gtggaagatg acgattttga 3120 tttttatgat gaattgtcaa aatggtggaa tgatactaag catgagctac cagattttga 3180 tgaattcaat tataccgttc cagttttaaa tattagtaat gaaattgaca gaattcaaca 3240 ggttattcag ggattaaatg attccctaat agaccttgaa acactctcaa ttctcaaaac 3300 ttattattaaa tggccttggt atgtgtggct tgccattgca ttccttacca ttatttttat 3360 tctggtactt tgttggatat tttcatgac cggttgttgc ggttgttgtt gtggatgctt 3420 tggtatcata ccgttaatga gtaagtgtgg taagaaatct tcttactaca cgacttttga 3480 taatgatgtg gtaacttaac aatacagacc taaaaagtct gtttaatgat taaaagtccc 3540 acatcttttc taatattatt aattcttctt tggtgttaaac ttgcattaag ttgttttaaa 3600 3660 ttacaatcta gacttctgct ttggcacagt ctagactaat gttagatttt gaagcaatta 3720 ttgaaactgg tcagcaaata actcaaaaa ttagtttcta tttacagcat atttcaaggg 3780 tgctaagtac tgaattatt gacccctttg aagtttgtgt ttacagagga ggtaattgtt 3840 gggagttaga gtcagctgac gagttttcag gtgatgacga atatattgag tagatcgctc 3900 gagaatcact agtgcggccg cctgcaggtc gaccatatgg gagagctccc aacgcgttgg 3960 atgcatagct tgagtattct atagtgtcac ctaaatagct tggcgtaatc atggtcatag 4020 ctgtttcctg tgtgaaattg ttatccgctc acaattccac aaacatacg agccggaagc 4080 ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc 4140 tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa 4200 cgcgcggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg 4260 ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg 4320 ttatccacag aatcaggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag 4380 gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac 4440 gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga 4500 taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt 4560 accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc 4620 tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc 4680 cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta 4740 agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat 4800 gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctcac tagagaaca 4860 gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct 4920 tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt 4980 acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct 5040 cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc 5100 acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa 5160 acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta 5220 tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc 5280 ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat 5340 ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta 5400 tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt 5460 aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt 5520 ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg 5580 ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc 5640 gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc 5700 gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg 5760 cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga 5820 actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta 5880 ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct 5940 tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag 6000 ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca atattattga 6060 agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat 6120 aaaaatag gggttccgg cacatttccc cgaaaagtgc cacctgatgc ggtgtgaaat 6180 accgcacaga tgcgtaagga gaaaataccg catcaggaaa ttgtaagcgt tatattttg 6240 ttaaaattcg cgttaaattt ttgttaaatc agctcatttt ttaaccaata ggccgaaatc 6300 ggcaaaatcc cttataaatc aaaagaatag accgagatag ggttgagtgt tgttccagtt 6360 tggaacaaga gtccactatt aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc 6420 tatcagggcg atggcccact acgtgaacca tcaccctaat caagtttttt ggggtcgagg 6480 tgccgtaaag cactaaatcg gaaccctaaa gggagccccc gatttagagc ttgacgggga 6540 aagccggcga acgtggcgag aaaggaaggg aagaaagcga aaggagcggg cgctagggcg 6600 ctggcaagtg tagcggtcac gctgcgcgta accaccacac ccgccgcgct taatgcgccg 6660 ctacagggcg cgtccattcg ccattcaggc tgcgcaactg ttgggaaggg cgatcggtgc 6720 gggcctcttc gctattacgc cagctggcga aagggggatg tgctgcaagg cgattaagtt 6780 gggtaacgcc agggttttcc cagtcacgac gttgtaaaac gacggccagt gaattgtaat 6840 acgactcact atagggcgaa ttgggcccga cgtcgcatgc tcccggccgc catggccgcg 6900 ggatt 6905 <210> 14 <211> 11320 <212> DNA <213> Artificial Sequence <220> <223> Plasmid Sequence <400> 14 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180 acdatagcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 240 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 300 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 360 tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt ggagatcggt acttcgcgaa 420 tgcgtcgaga tgagctctaa tacgactcac tatagggact taagtgtgat ataaatatat 480 atcatacata ctagccttgt gctagatttc caacttaaca aaacggactt aaatacctac 540 agttggtccc tataggtgtt ccattgcagt gcactttagt gccctggatg gcacctggcc 600 acctgtcagg tttttgttat taaaataata ttgttgctgg tatcactgct tgttttgccg 660 tgtctcactt tatacatccg ttgcttgggc tacctagtat ccagcgtcct actggcgttg 720 tggtcggttc gagtgcgaag aacctctggt tcatctagcg gtacgcgggt gtgtggaagt 780 agcgcttcag acgtaccggt tctgttgcgt gaaatacggg gtcacctccc cccacatacc 840 tctaagggct tttgagccta gcgttgggct acgttctcgc acaaggtcgg ctaacggcg 900 tttgtagggg gtagtgccaa acaacccctg aggtgacagg ttctggtggt gtttcgaaaa 960 caacaatgtg tgtgccgcat aatatgcgag taatgcattt tggagcagga agtgacaaag 1020 gagtggcccc aggtagcgct gttcttaggc agtggcttcc cgaaggtaca ctccttgtcg 1080 ataatgatat tggatatt gtatctgatg cacatgtctc tgtgctttca gattgcaata 1140 aatgtaaaac agagcacaag tttgatcttg tgatatctga tatgtataca gataatgatt 1200 caaagagaaa gcatgaaggc gttgtagcca ataacggcaa tgatgacgtc ttcatatacc 1260 tttcaaactt tcttcgtaac aatttagctc tgggaggcag ttttgccgta aaagtaacag 1320 agacaagttg gcatgagagt ttatatgaca ttgcacagga ttgtgcatgg tggacaatgt 1380 tttgtacagc agtgaatgct tcttcgtcag aagcattctt gattggtgtt aattattgg 1440 gtgcaagtga aaggttaga gttagtgta aaaccctgca cgcaattat atttttgga 1500 ggaattgtaa ttatttaca acttcagcct atagtatatt tgacgttgct aagtttgatt 1560 tgaaatttaa agcaacgcca gttgtgaatt tgaaaactga acaaagaca gacttagtct 1620 ttaatttaat taagtgtgt aagttactgg taagagatgt tggacaacc gcttttacta 1680 gtgactcttt ggtagcact atgtagtgct ttgctctg ataatatac ttacgtttac 1740 tactaccaaa gtgccttag gcctggtcca ggttggcacc tatatggggg tgcttatgca 1800 gtagataggg ttttaatga aaccaacaat gcaggcagtg catctgattg cactgctggt 1860 actttttag aaagccataa tatttctgct tcttctgtag ccatgacagt accacataat 1920 ggtagtctt gtcagcttc acaatttgt acagctcatt gtactctc agactttaca 1980 gtgttcgtta cgcattgttt taaaaatcaa ctcggtagtt gtcccttgac aggtatgatt 2040 cctcagaatc atattcgtat ttctgctatg agagatggag tttgtttta taacttaaca 2100 gttagcgtat ctaaataccc tegttaaa tcgcttcaat gtgttagcaa ttctacatct 2160 gtctatgtaa atggtgacct tgttttcact tctaatgaaa cttcttacgt tacgggtgca 2220 ggcgtttatt ttaaaagtgg tgggcctgta acttataaag tttgaaaga agttaaagcc 2280 ctagcctact ttattaatgg taccgcacaa gaggttattt tatgtgataa ctcacctaga 2340 ggtttgcttg catgtcagta taacactggt aatttttcag atggattcta cccttttact 2400 aatcattctt tagttaagga taggtttatt gtatatcgag aaagtagcac taacactact 2460 ttaaagttaa ctaatttcag ttttactaat gtaagtaatg cttctcctaa ttcaggtggc 2520 gttgatactt tccaattata tcaaacaagt actgctcagg atggttatta taattttaat 2580 ttatcatttc tgagtagttt tgtgtataaa ccatctgatt ttatgtatgg gtcataccac 2640 ccacattgta agtttagacc agagaatatt aataatggct tatggtttaa ttcattatct 2700 gtgtcactta cttacggacc cattcaaggt ggttgtaagc aatctgtttt tagtaataga 2760 gcaacttgtt gctatgctta ttcttatcaa gggcctagta gatgtaaggg tgtttataga 2820 ggggagctaa cgcaatactt tgaatgtgga cttctagttt acgtaactaa gagtgatggc 2880 tctcgtatac aaactagaag tgaaccactg gtgttaactc aatataatta taacaacatt 2940 actttaaata agtgtgttga gtataatata tatggtaggg ttggtcaagg ttttattact 3000 aatgtaactg aagcaactgc taattatagt tatctagcag atggtggttt agctatttta 3060 gatacctcag gagccataga catatttgtt gttcaaggtg catatggtct taattattat 3120 aaggttaatc cctgtgaaga tgttaaccaa cagtttgtag tgtctggtgg caacttagtt 3180 ggcattctta catctcataa tgaaacaggt tctgaatcta ttgagaacca gttttacatc 3240 aaactcacta acggaacacg tcgctctaga cgttctgtta ctgggaatgt tacaaattgc 3300 ccttatgtta gttatggcaa gttttgtata aaaccagatg gttctttatc tataatagta 3360 ccacaagaat tagaacagtt tgtggcgcct ttattcaatg ttactgagca tgtgctcata 3420 cctgatagtt ttaatttaac tgtcacagat gagtacatac aaactcgtat ggataaggtt 3480 caaattattt gccttcagta tgtttgtggt aattctattg aatgcagaaa gttgtttcag 3540 cagtatggac ctgtttgtga tatatattg tctgttgtaa atggtgtagg tcaaagagag 3600 gatatggaac ttttaagttt ctattcttct actaaaccta gtggttacaa tacaccaatt 3660 tttaataatg ttagcactgg tgactttaat atttcgctcc tactaacacc acctaatagt 3720 cctactgggc gctcttttat tgaagatctt cctttacaa gtgtagaatc tgttggatta 3780 ccaactgatg aagagtataa aaagtgtaca gcaggacctt taggttttgt taaagacctt 3840 gttgtgcta gagagtataa tggtttgctc gttctgcctc ctattattac tgcggaaatg 3900 caaaccatgt atactagttc tttagtagcc tctatggctt taggtggcat tactgcagct 3960 ggtgctatac cttttgctac acaactgcag gccagaatta accatttggg tattactaat 4020 tctcttttgt tgaaaaacca agaaaaaatt gctgcttcct ttaataaggc catcggtcat 4080 atgcaggaag ggtttaaaag tacttctcta gcattacaac agattcaaga tgttgttaat 4140 aaacagagtt ctattcttac agagactatg caatcactta ataaaaattt tggtgctatt 4200 tcctctgtaa ttcaagacat ttaccagcaa ctagatgcta ttcaggcaga tgctcatgtt 4260 gatcgtctta ttacaggtag actctcttca ctatctgttt tagcttctgc taaacaggca 4320 gagtatcata gagtgtcaca acagcgtgag ttggccactc agaaaattaa tgagtgtgtt 4380 aagtctcagt ctaataggta ttcattttgt ggtaatggta gacatgttct aaccatacca 4440 cagaatgcac ccaatggcat agtgtttata cactttacat acactccaga gagttttgtt 4500 aatgttacgg caatagtagg gttttgcgta aacccagcta atgctagtca ttatgcaata 4560 gtgcctgtta atggcagggg tgtttttata gaagttaatg gtagttacta tatcactgct 4620 cgtgatatgt atatgccaag agatattact gcaggagaca tagtcacttt gacttcttgt 4680 caagcaaact atgttaatgt aaataaaacc gtcattaaca cttttgtgga agatgacgat 4740 tttgattttt atgatgaatt gtcaaaatgg tggaatgata ctaagcatga gctaccagat 4800 tttgatgaat tcaattatac cgttccagtt ttaaatatta gtaatgaaat tgacagaatt 4860 caacaggtta ttcagggatt aaatgattcc ctaatagacc ttgaaacact ctcaattctc 4920 aaaacttata ttaaatggcc ttggtatgtg tggcttgcca ttgcattcct taccattatt 4980 tttattctgg tactttgttg gatatttttc atgaccggtt gttgcggttg ttgttgtgga 5040 tgctttggta tcataccgtt aatgagtaag tgtggtaaga aatcttctta ctacacgact 5100 tttgataatg atgtggtaac ttaacaatac agacctaaaa agtctgttta atgattaaaa 5160 gtcccacatc ttttctaata ttattaattc ttctttggtg taaacttgca ttaagttgtt 5220 ttaaagagtg tgttataaca ctccagcaac tagtacaaat tttactccaa attattaata 5280 gtaacttaca atctagactt ctgctttggc acagtctaga ctaatgttag attttgaagc 5340 aattattgaa actggtcagc aaataactca acaaattagt ttctatttac agcatatttc 5400 aagggtgcta agtactgaat tatttgaccc ctttgaagtt tgtgtttaca gaggaggtaa 5460 ttgttgggag ttagagtcag ctgacgagtt ttcaggtgat gacgaatata ttgagtagat 5520 cgctcgagga gaacggaagt tttctaacag cggtttacgt gtttttagga tttttagcac 5580 tttatctact aggtagagcg cttcaagctt ttgtacaagc ggctgacgct tgttgtcttt 5640 tttggtatac atgggtagta gttcctggag ccaagggcac agcctttgtt tataatcata 5700 catatggtaa aaaacttaac aaaccggagt tagaaacggt tattgttaac gaatttccaa 5760 aaaacggttg gaatatgga taataccatc aattgtactc ttggtactga acagcagtt 5820 cagcttttta aggaatataa tctgtttgta actgcattcc tgttgttttt aaccatacta 5880 cttcagtag gatacgcaac taggagcaag gttattaca tactgaaat gatagtgtta 5940 tggtgctttt ggccccttaa cattgcagta ggtgtaatct catgtatata cccaccaac 6000 acaggaggtc ttgtcgcagc gataatttt acagtgttg cgtgtctttc ttttaggt 6060 tattggatcc agagtattag actttag cggtgcaggt catggtggtc atttacccc 6120 gatctaatg ccgtaggttc atactccta actaatggtc aacaatgtaa tttgctata 6180 gagagtgtgc cgatggtgct ttctccttatt ataagaatg gtgctcttta ttgcgaggt 6240 cagtggcttg ctaaatgtga accagaccac ttgcctag atatatttgt atgcacaccg 6300 gatagacgta atatctatcg tattggtgca aaatactg gtgaccaag cggagtag 6360 aaaaggttg ccacattgt ctatgcaaag cagtcagtag atactggcga gctagaaagt 6420 gtgtcagcag taggagg tctttacaca taaatgtgtg tgtgtagaga gtatttaaaa 6480 ttatctttg acagtgcctc cgttttaaga gcgcggaaga gtattatttt tgaggatatt 6540 aatataaatc ctctttgttt catactctcc tttcaggagt tattatttaa aaaacagttt 6600 ttccactctt ttgtgccaaa aacaattgtt gttaatggtg taacctttca ggtagacaat 6660 ggaaaagtct actacgaagg aagaccaatt ttccaaaaag gttgttgtag tttgtggtcc 6720 aattataaga aagattagaa taattaaacc acctacaaca cttattttta caaatggcgt 6780 tttaggttac aaacgcttaa caaatacgga tgatgaaatg gctgactagt tttggaagag 6840 ctttcatctc ctgttataaa tccctattac taactcaatt aagagtatta gataggttaa 6900 ttttagatca cggacccaag cgcacatta cgtgtgctag gcgagtgctt ttagttcaat 6960 tagatttagt ttatagggttg gcttatacgc ccacccaatc gctggtatga samsaagtaa 7020 agataatcct tttcgcggag caatagcaag aaaagcgcga atttatctga gaggaggatt 7080 agattgtgtt tactttctta acaaagcagg acaagcagag ccttgtcccg cgtgtacctc 7140 cctagtattc caaggggaaaa cttgtgagga acactattat aataacaatc ttttgtcatg 7200 gcaagcggta aggcaactgg aaagacagac gccccagcgc cagtcatcaa actaggagga 7260 ccaaagccac ctaaagttgg ttcttctgga aatgcatcat ggtttcaacc gataaaggcc 7320 aagaagctaa attcacctgt gcctaaattt gacggtagtg gtgttcctga aaatgaaaat 7380 ctcaagtcaa gccagcaaca tggatactgg agacgccaac acaggtttaa gcctggcaaa 7440 ggtggaagaa aaccagtccc tgatgcttgg tacttttact acactggaac aggaccggcc 7500 gccgacctga attggggtga aactcaagat ggtatagtgt gggttgctgc aaagggtgct 7560 gatactaaat ctagatcaaa ccagggtaca agggatcctg ataagtttga ccaataccca 7620 ctacgattct cagatggagg accggatggt aatttccgtt gggacttcat accaataaat 7680 cgtggtagga gtgggagatc aacagcagct tcatcagcag catctagtag agcaccatct 7740 cgtgaggggt cacgtggacg tagaagcgga gttgaagatg atcttatagc tcgcgcagca 7800 aagattatac aggaccagca aaagaagggt gcgcgcatta ccaaggctaa ggctgatgaa 7860 atggctcatc gccgctattg caagcgcact atcccacctg gttataaggt tgagcaagta 7920 tttggtcccc gtactaaagg taggaaagga aattttggtg atgacaagat gaatgaggaa 7980 ggtgttaagg atgggcgtgt tacggcaatg ctcaacctag tccctagcag tcatgcttgt 8040 ctttttggaa gtagggtgac gcccaaactg cagccagatg gttttcacct gagatttgaa 8100 tttactactg tggtgtcacg tgatgatccg cagtttgata attatgtgaa aatttgtgat 8160 cagtgtgtcg atggtgtagg gacgcgtcca areacgatg aatcgagacc aaagtcacgc 8220 ccaaattcaa gacctgcaac tagaggaaat tctccagcgc cgagacaaca gcgcccaaag 8280 aaggagaaaa agcccaagaa gcaggatgat gaagtagata aggcattgac ctcagatgag 8340 gaggaaca atgcacagct ggaatttgat gatgaaccca aggtgattaa ctggggggac 8400 tctgcactag gtgaaaatga actttgatta acataatgga cttgctgcat ttgctgtcac 8460 attttgttaa atattatttt tgtgttttac tatcaattat tacaggtatt gattgtgatt 8520 atgttcaata cttaagcttc ttctggttgc tttttgcttg ttgtattgtt gctgtgcttt 8580 ttattattgt gattctcatt agtttgcttt atcgtagaaa ttcaatagta agagttaagg 8640 aagataggca tgtagcttag cacctacatg tctatcgcca gggaaatgtc taatctgtct 8700 acttagtagc ctggaaacga acggtagacc cttagatttt aatttagttt aatttttagt 8760 ttagtttaag ttagtttaga gtaggtataa agatgccagt gccggggcca cgcgtagtac 8820 gaccgagggt acagcactag gacgcccact aggggaagag ctaaatttta gtttaagtta 8880 agtttaattg gctaaatata gttaaaattt ataggctagt atagagttag agcaaaaaaa 8940 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 9000 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaagcggccg catcggatgc cgggaccgac 9060 gagtgcagag gcgtgcaagc gagcttggcg taatcatggt catagctgtt tcctgtgtga 9120 aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc 9180 tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc 9240 cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc 9300 ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt 9360 cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca 9420 ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa 9480 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat 9540 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc 9600 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc 9660 gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt 9720 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac 9780 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg 9840 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca 9900 gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc 9960 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa 10020 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa 10080 ggatctcaag aagatccttt gatctttct acggggtctg acgctcagtg gaacgaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc 10380 agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc 10620 agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc 10740 atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc 10860 tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc 10920 atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc 10980 agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc 11040 gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca 11100 cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt 11160 tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt 11220 ccgcgcacat ttccccgaaa agtgccacct gacgtctaag aaaccattat tatcatgaca 11280 ttaacctata aaaataggcg tatcacgagg ccctttcgtc 11320 <210> 15 <211> 6905 <212> DNA <213> Artificial Sequence <220> <223> Plasmid Sequence <400> 15 ttaattaagt gtggtaagtt actggtaaga gatgttggac aaaccgcttt tactagtgac 60 tctttggtat gcactatgta gtgctttgct ctatgataat aatacttacg tttactacta 120 ccaaagtgcc tttaggcctg gtccaggttg gcacctatat gggggtgctt atgcagtaga 180 tagggttttt aatgaaacca acaatgcagg cagtgcatct gattgcactg ctggtacttt 240 ttatgaaagc cataatattt ctgcttcttc tgtagccatg acagtaccac ataatggtat 300 gtcttggtca gcttcacaat tttgtacagc tcattgtaac ttctcagact ttacagtgtt 360 cgttacgcat tgttttaaaa atcaactcgg tagttgtccc ttgacaggta tgattcctca 420 gaatcatatt cgtatttctg ctatgagaga tggagttttg ttttataact taacagttag 480 cgtatctaaa taccctagat ttaaatcgct tcaatgtgtt agcaattcta catctgtcta 540 tgtaaatggt gaccttgttt tcacttctaa tgaaacttct tacgttacgg gtgcaggcgt 600 ttattttaaa agtggtgggc ctgtaactta taaagttatg aaagaagtta aagccctagc 660 ctactttatt aatggtaccg cacaagaggt tattttatgt gataactcac ctagaggttt 720 gcttgcatgt cagtataaca ctggtaattt ttcagatgga ttctaccctt ttactaatca 780 ttctttagtt aaggataggt ttattgtata tcgagaaagt agcactaca ctactttaaa 840 gttaactaat ttcagtttta ctaatgtaag taatgctct cctaattcag gtggcgttga 900 tactttccaa ttatatcaa caagtactgc tcaggatggt tattatatt ttaatttatc 960 atttctgagt agttttgtgt aaaccatc tgattttag tatgggtcat accaccaca 1020 ttgtaagtttt agaccagaga atattaataa tggcttatgg tttattcat tatctgtgtc 1080 acttacttac ggacccattc aaggtggttg taagcaatct gttttagta atagagcac 1140 ttgttgctat gcttattctt atcaagggcc tagtagatgt aagggtgtttt atagagggga 1200 gctaacgcaa tactttgaat gtggacttct agtttacgta actaagagtg atggctctcg 1260 tatacaact agaagtgaac cactggtgtt aactcaat attackaca acattacttt 1320 aaataagtgt gttgagtata atatatatgg taggttggt caggtttta ttactaatgt 1380 aactgaagca actgctatt atagttactct agcagatgt gttttagcta ttttagatac 1440 ctcaggagcc atagacatat tgttgttca aggtgcatat ggtctttatt attaaggt 1500 taatccctgt gaagatgtta accaacagtt tgtagtgtct ggtggcaact tagttggcat 1560 tcttacatct cataatgaaa caggttctga atctattgag aaccagtttt acatcaaact 1620 cactaacgga acacgtcgct ctagacgttc tgttactggg aatgttacaa attgccctta 1680 tgttagttat ggcaagtttt gtataaaacc agatggttct ttatctataa tagtaccaca 1740 agaattagaa cagtttgtgg cgcctttatt caatgttact gagcatgtgc tcatacctga 1800 tagttttaat ttaactgtca cagatgagta catacaaact cgtatggata aggttcaaat 1860 tatttgcctt cagtatgttt gtggtaattc tattgaatgc agaaagttgt ttcagcagta 1920 tggacctgtt tgtgataata tattgtctgt tgtaaatggt gtaggtcaaa gagaggatat 1980 ggaactttta agtttctatt cttctactaa acctagtggt tacaatacac caatttttaa 2040 taatgttagc actggtgact ttaatattc gctcctacta acaccaccta atagtcctac 2100 tgggcgctct tttattgaag atcttctctt tacaagtgta gaatctgttg gattaccaac 2160 tgatgaagag tataaaaagt gtacagcagg acctttaggt tttgttaaag accttgtttg 2220 tgctagagag tataatggtt tgctcgttct gcctcctatt attactgcgg aaatgcaaac 2280 catgtatact agttctttag tagcctctat ggctttaggt ggcattactg cagctggtgc 2340 tatacctttt gctacacaac tgcaggccag aattaaccat ttgggtatta ctaattctct 2400 tttgttgaaa aaccaagaaa aaattgctgc ttcctttaat aaggccatcg gtcatatgca 2460 ggaagggttt aaaagtactt ctctagcatt acaacagatt caagatgttg ttaataaaca 2520 gagttctatt cttacagaga ctatgcaatc acttaataaa aattttggtg ctatttcctc 2580 tgtaattcaa gacatttacc agcaactaga tgctattcag gcagatgctc atgttgatcg 2640 tcttattaca ggtagactct cttcactatc tgttttagct tctgctaaac aggcagagta 2700 tcatagagtg tcacaacagc gtgagttggc cactcagaaa attaatgagt gtgttaagtc 2760 tcagtctaat aggtattcat tttgtggtaa tggtagacat gttctaacca taccacagaa 2820 tgcacccaat ggcatagtgt ttatacactt tacatacact ccagagagtt ttgttaatgt 2880 tacggcaata gtagggtttt gcgtaaaccc agctaatgct agtcattatg caatagtgcc 2940 tgttaatggc aggggtgttt ttatagaagt taatggtagt tactatatca ctgctcgtga 3000 tatgtatatg ccaagagata ttactgcagg agacatagtc actttgactt cttgtcaagc 3060 aaactatgtt aatgtaaata aaaccgtcat taacactttt gtggaagatg acgattttga 3120 tttttatgat gaattgtcaa aatggtggaa tgatactaag catgagctac cagattttga 3180 tgaattcaat tataccgttc cagttttaaa tattagtaat gaaattgaca gaattcaaca 3240 ggttattcag ggattaaatg attccctaat agaccttgaa acactctcaa ttctcaaaac 3300 ttattattaaa tggccttggt atgtgtggct tgccattgca ttccttacca ttatttttat 3360 tctggtactt tgttggatat tttcatgac cggttgttgc ggttgttgtt gtggatgctt 3420 tggtatcata ccgttaatga gtaagtgtgg taagaaatct tcttactaca cgacttttga 3480 taatgatgtg gtaacttaac aatacagacc taaaaagtct gtttaatgat taaaagtccc 3540 acatctttc tatattatt aattcttct tggtgtaaac ttgcattaag ttgttttaaa 3600 gagtgtgtta taacactcca gcaactagta caaattttac tccaaattat tatagtaac 3660 ttacaatcta gacttctgct ttggcacagt ctagactaat gttagatttt gaagcaatta 3720 ttgaaactgg tcagcaaata actcaacaaa ttagtttcta tttacagcat atttcaaggg 3780 tgctaagtac tgaattattt gacccctttg aagtttgtgt ttacagagga ggtaattgtt 3840 gggagttaga gtcagctgac gagttttcag gtgatgacga atatattgag tagatcgctc 3900 gagaatcact agtgcggccg cctgcaggtc gaccatatgg gagagctccc aacgcgttgg 3960 atgcatagct tgagtattct atagtgtcac ctaaatagct tggcgtaatc atggtcatag 4020 ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc 4080 ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc 4140 tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa 4200 cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg 4260 ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg 4320 ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag 4380 gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac 4440 gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga 4500 taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt 4560 accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc 4620 tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc 4680 cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta 4740 agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat 4800 gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctcac tagagaaca 4860 gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct 4920 tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt 4980 acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct 5040 cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc 5100 acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa 5160 acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta 5220 tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc 5280 ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat 5340 ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta 5400 tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt 5460 aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt 5520 ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg 5580 ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc 5640 gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc 5700 gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg 5760 cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga 5820 actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta 5880 ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct 5940 tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag 6000 ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca atattattga 6060 agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat 6120 aaaaatag gggttccgg cacatttccc cgaaaagtgc cacctgatgc ggtgtgaaat 6180 accgcacaga tgcgtaagga gaaaataccg catcaggaaa ttgtaagcgt tatattttg 6240 ttaaaattcg cgttaaattt ttgttaaatc agctcatttt ttaaccaata ggccgaaatc 6300 ggcaaaatcc cttataaatc aaaagaatag accgagatag ggttgagtgt tgttccagtt 6360 tggaacaaga gtccactatt aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc 6420 tatcaggcg atggcccact acgtgaacca tcaccctaat caagtttttt ggggtcgagg 6480 tgccgtaaag cactaaatcg gaaccctaaa gggagccccc gatttagagc ttgacgggga 6540 aagccggcga acgtggcgag aaaggaaggg aagaaagcga aaggagcggg cgctagggcg 6600 ctggcaagtg tagcggtcac gctgcgcgta accaccacac ccgccgcgct taatgcgccg 6660 ctacagggcg cgtccattcg ccattcaggc tgcgcaactg ttgggaaggg cgatcggtgc 6720 gggcctcttc gctattacgc cagctggcga aagggggatg tgctgcaagg cgattaagtt 6780 gggtaacgcc agggttttcc cagtcacgac gttgtaaaac gacggccagt gaattgtaat 6840 acgactcact atagggcgaa ttgggcccga cgtcgcatgc tcccggccgc catggccgcg 6900 ggatt 6905 <210> 16 <211> 47 <212> DNA <213> Artificial sequence <220> <223> Primer sequence <400> 16 ttaattaagt gtggtaagtt gcttgtaaga gatgttggta acacctc 47 <210> 17 <211> 47 <212> DNA <213> Artificial sequence[[ID=3s7]] <220> <223> Primer sequence <400> 17 ctcgagcgac ttattcaata aattcatcat taaacagact ttttagg 47 <210> 18 <211> 39 <212> DNA <213> Artificial sequence <220> <223> Primer sequence <400> 18 ccatacaagc aaatgctcac gtggatcgtc ttataactg 39 <210> 19 <211> 19 <212> DNA <213> Artificial sequence <220> <223> Primer sequence <400> 19 tgctgcttcc tttaataag 19 <210> 20 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Primer sequence <400> 20 aacactatac cattaggtgc 20 <210> twenty one <211> 52 <212> DNA <213> Artificial sequence <220> <223> Primer sequence <400> twenty one cagagcacaa gtttgatctt gtgatatctg atatgtatac agacaatgat tc 52 <210> twenty two <211> 32 <212> DNA <213> Artificial sequence <220> <223> Primer sequence <400> twenty two gtgttaccaa catctcttac cagtaactta cc 32 <210> twenty three <211> 35 <212> DNA <213> Artificial sequence <220> <223> Primer sequence <400> twenty three ttactggtaa gagatgttgg taacacctct tttac 35 <210> twenty four <211> 35 <212> DNA <213> Artificial sequence <220> <223> Primer sequence <400> twenty four ggactttgga tcattaaaca gactttttag gtctg 35 <210> 25 <211> 32 <212> DNA <213> Artificial sequence <220> <223> Primer sequence <400> 25 aaagtctgtt taatgatcca aagtcccact ag 32 <210> 26 <211> 48 <212> DNA <213> Artificial sequence <220> <223> Primer sequence <400> 26 cttaactcct ggaattacta accacgtgta ccaaaataaa caacaagc 48 <210> 27 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Primer sequence <400> 27 tgacttggtt tgaagatggc 20 <210> 28 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Primer sequence <400> 28 ccccatgtaa atgccaacca 20 <210> 29 <211> 39 <212> DNA <213> Artificial sequence <220> <223> Primer sequence <400> 29 ctattcaggc agatgctcat gttgatcgtc ttattacag 39 <210> 30 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Primer sequence <400> 30 tcagcgtgga catgtggtta 20 <210> 31 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Primer sequence <400> 31 ccccatatag gtgccaacct 20 Attached Figure Description

[0534] Figure 1 .H52 rIBV S Q865H in Three blind passages were performed in the cells. For all three passages, replication was evaluated by IBV-specific RT-qPCR at 0 and 72 hours post-infection.

[0535] Figure 2 Compared with uninfected negative controls, those infected with H52 rIBV S Q865H... Immunofluorescence staining of cells.

[0536] Figure 3Immunofluorescence staining of BHK cells infected with H52 rIBV S Q865H at passage 3 compared to uninfected negative controls.

[0537] Figure 4 Immunofluorescence staining of BHK cells infected with H52 rIBV SQ865H at passage 3 compared to uninfected negative controls and cells infected with wild-type H52 rIBV.

[0538] Sequence Overview

[0539] SEQ ID NO:1IBV H52 spike protein

[0540] SEQ ID NO:2 IBV H52 spike protein with Q865H mutation

[0541] SEQ ID NO:3 IBV CR88 spike with Q867H mutation

[0542] SEQ ID NO:4 IBV QX spike protein with Q868H mutation

[0543] SEQ ID NO:5 IBV Q1 spike protein with Q869H mutation

[0544] SEQ ID NO:6 IBV Var 2 spike protein with Q868H mutation

[0545] SEQ ID NO:7 IBV BR-I spike protein with Q872H mutation

[0546] SEQ ID NO:8 IBVArk spike protein with Q871H mutation

[0547] SEQ ID NO:9pUC57-s H52 rIBV S Q865H donor plasmid

[0548] SEQ ID NO:10pUC57-s H52 rIBV S donor plasmid

[0549] SEQ ID NO:11IBV CR88 spike protein

[0550] SEQ ID NO:12pUC57-s CR88 mIBV donor plasmid

[0551] SEQ ID NO:13pGEM-T CR88 S

[0552] SEQ ID NO:14pUC57-s CR88 rIBV S Q867H donor plasmid

[0553] SEQ ID NO:15pGEM-T CR88 S Q867H

[0554] SEQ ID NO:16-31 primers

[0555] Example

[0556] The following embodiments illustrate specific implementations of the present invention. These embodiments are merely illustrative and should not be construed as limiting the scope or basic principles of the invention.

[0557] Example 1: Recombinant IBV in which amino acid 865 of the spike protein is mutated to histidine. H52 generation

[0558] For the generation of recombinant IBV, a method for targeted RNA recombination, as described by van Beurden et al. (Virol J. 2017; 14(1):109), is applied.

[0559] donor plasmid construction

[0560] The H52 spike protein sequence (SEQ ID NO:1) was amplified by PCR using primers 1044 and 1045 (Table 1) and cloned into... Within the Promega vector system, plasmid pGEM-T IBV H52 spikes were generated. Following the kit protocol, a mutation from glutamine to histidine was introduced at position 865 of the S2 subunit (SEQ ID NO:2) of the H52 spike using the QuikChange Multi Site-Directed Mutagenesis Kit (Agilent Technologies) along with primer PO2079 (Table 1). Positive plasmids were identified post-transformation by restriction digestion with NcoI and NsiI prepared from the cloned bacterial culture. To identify plasmids with the targeting mutation, Sanger sequencing of plasmids with the expected restriction pattern was performed using primers PO617 and PO634 (Table 1).

[0561] Table 1: Primers used for cloning, site-directed mutagenesis, and characterization of the pGEM-T IBV H52 spike plasmid.

[0562]

[0563]

[0564] To generate the H52 donor plasmid pUC57-s H52 rIBV S Q865H (SEQ ID NO:9), the NEBuilder HiFi DNA Assembly Cloning Kit (NEB) was used according to the kit protocol. For Gibson assembly, the H52 S Q865H sequence was amplified from the pGEM-T IBV H52 S Q865H plasmid via PCR. Further, two PCRs were performed to generate flanking sequences of the spike protein, including EcoRV and BlpI restriction sites, against the background of the IBV H52 viral genome (Table 2). For this purpose, the pUC57-s IBV-5-1b-S-SIR-3T donor plasmid described by van Beurden et al., hereinafter referred to as pUC57-sH52 rIBV donor plasmid (SEQ ID NO:10), was used as a template. The correct size and purity of the PCR products were determined by agarose gel electrophoresis, and the PCR products were subsequently purified using the QIA Rapid PCR Purification Kit (QIAGEN). In parallel, the pUC57-s H52 rIBV donor plasmid was digested with EcoRV and BlpI to obtain a donor plasmid backbone sequence of approximately 7000 base pairs for Gibson assembly. Positive plasmids were identified by restriction digestion with EcoRV and XbaI. Positive plasmids were sequences with primers PO619 and PO634 (Table 1) to confirm the presence of the Q865H mutation in the spike S2 ​​subunit.

[0565] Table 2: Setup for generating PCR products to obtain pUC57-s H52 rIBVS Q865H donor plasmid assembled with Gibson HiFi.

[0566]

[0567] Rescue of Targeted RNA Recombination and Recombinant IBV

[0568] As described by van Beurden et al. (Virol J. 2017; 14(1):109), H52 murine (m)IBV helper virus and recombinant IBV were generated. In short, for the generation of H52 rIBV S Q865H, LR7 cells were infected with H52 mIBV and electroporated with an in vitro transcript generated from the pUC57-s H52 S Q865H donor plasmid (SEQ ID NO:9), and subsequently injected into 8-day-old SPF embryo-containing eggs (VALO BioMedia). After incubation for up to 8 days, the embryos were analyzed using MagMAX. TMCore Nucleic Acid Purification Kit (ThermoFisher) and KingFisher TM DuoPrime Purification System (ThermoFisher), and by using Platinum TM SuperScript for Taq DNAPolymerase TM III. The One-Step RT-PCR System (ThermoFisher) was used to rescue recombinant IBV after RNA isolation, and allantoic fluid from all eggs was analyzed separately. Primers PO765 and PO1324 (Table 3) binding to H52 IBV 1ab and H52 IBV S spikes were used to distinguish recombinant IBV from mIBV. ​​Positive allantoic fluid from one egg inoculated with infected and electroporated LR7 cells was used for endpoint dilution in 8-day-old SPF embryo-containing eggs. Nucleic acid isolation and sample analysis were performed as described above. The same procedure was applied to the second endpoint dilution procedure. Subsequently, a positive allantoic fluid was used for propagation in 10-day-old SPF embryo-cont...

Claims

1. An infectious bronchitis virus spike protein with histidine at position 865, wherein the spike protein is not from infectious bronchitis virus strain M41, and wherein the amino acid sequence of SEQ ID NO:1 is used to determine the position number in the spike protein; wherein the spike protein is from the following infectious bronchitis virus: H120 strain or H52 strain.

2. An infectious bronchitis virus spike protein with histidine at position 867, wherein the spike protein is not from infectious bronchitis virus strain M41, and wherein the amino acid sequence of SEQ ID NO:11 is used to determine the position number in the spike protein; wherein the spike protein is from infectious bronchitis virus CR88.

3. The infectious bronchitis virus spike protein of any one of claims 1 to 2, wherein the histidine at amino acid position 865 or 867 results in extended cell tropism or tissue tropism of the infectious bronchitis virus.

4. The infectious bronchitis virus spike protein of any one of claims 1 to 2, wherein the infectious bronchitis virus infects and / or replicates in at least one cell line selected from the group consisting of DF-1, PBS-12SF, BHK21, HEK 293T, Vero, MA104, RK13, LMH, MDCK, MDBK, PK15, PK2A, SF9, SF21, and SF+.

5. The infectious bronchitis virus spike protein of any one of claims 1 to 2, wherein the infectious bronchitis virus spike protein consists of: The amino acid sequence as shown in SEQ ID NO: 2 or 3.

6. A nucleic acid molecule encoding the spike protein of any one of claims 1 to 5.

7. A plasmid comprising the nucleic acid molecule of claim 6.

8. A viral particle comprising the spike protein of any one of claims 1 to 5.

9. An infectious bronchitis virus comprising the spike protein of any one of claims 1 to 5.

10. The infectious bronchitis virus of claim 9, wherein the infectious bronchitis virus is attenuated.

11. A cell comprising: the plasmid of claim 7, or the viral particle of claim 8, or the infectious bronchitis virus of claim 9.

12. An immunogenic composition comprising: the spike protein of any one of claims 1 to 5, or the viral particle of claim 8, or the infectious bronchitis virus of claim 9.

13. A method for producing an infectious bronchitis virus having extended cell tropism or tissue tropism, comprising using the infectious bronchitis virus spike protein of any one of claims 1 to 5.

14. A method for culturing infectious bronchitis virus in cell culture or tissue culture, comprising using the infectious bronchitis virus spike protein of any one of claims 1 to 5.

15. Use of the following substances in the preparation of an immunogenic composition for preventing clinical signs caused by infectious bronchitis virus in a subject in need thereof: the spike protein of any one of claims 1 to 5, or the viral particle of claim 8, or the infectious bronchitis virus of claim 9.

16. Use of the immunogenic composition of claim 12 in the preparation of a kit for preventing clinical signs caused by infectious bronchitis virus in a subject in need thereof.

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