H52 IBV vaccine with heterologous spike protein

Abstract

The present invention relates to H52IBV (infectious bronchitis virus), which encodes a heterologous S (spike) protein or a fragment thereof. Further, the present invention relates to an immunogenic composition comprising the H52IBV encoding the heterologous S (spike) protein or a fragment thereof. Furthermore, the present invention relates to a method for immunizing a subject, comprising administering the immunogenic composition of the present invention to the subject. In addition, the present invention relates to a method for treating or preventing clinical signs caused by IBV in a subject in need thereof, the method comprising administering a therapeutically effective amount of an immunogenic composition according to the present invention to the subject.

Description

[0001] Sequence Listing

[0002] This application contains a Sequence Listing according to 37 CFR 1.821-1.825. The Sequence Listing accompanying this application is incorporated herein by reference in its entirety. Background of the Invention

[0004] Avian coronavirus infectious bronchitis virus (IBV) is a prototype gamma coronavirus of the family Coronaviridae of the order Nidovirales. Infectious bronchitis virus primarily infects the upper respiratory epithelium of chickens, causing respiratory disease, often complicated by secondary bacterial pathogen infection (Cook et al. 2012. Avian Pathol. 41: 239-250). Some IBV strains also affect the renal tubules, oviducts, and parts of the gastrointestinal tract, causing pathological lesions and clinical signs in these organ systems. The virus is widely present in commercial poultry and backyard poultry. Due to its high genomic variability, IBV is divided into multiple genotypes, serotypes, and protective types. IBV is currently considered to be one of the most economically relevant viral pathogens in the poultry industry.

[0005] Infectious bronchitis virus is an enveloped virus with a 27.6kb positive 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 called gene 1), divided into two open reading frames 1a and 1b, which encode 15 non-structural proteins involved in RNA replication, editing and transcription. The last third of the viral genome encodes structural proteins: spike protein (S, encoded by gene 2), envelope protein (E, encoded by gene 3c), membrane protein (M, encoded by gene 4), nucleocapsid protein (N, encoded by gene 6). Proteins S, E and M are part of the viral envelope, while protein N forms the ribonucleoprotein core together with viral RNA. The coronavirus spike protein determines the host species tropism (Kuo et al.2000.J.Virol.74:1393-1406). It is a dimeric or trimeric transmembrane protein that is proteolytically broken down into two subunits, S1 and S2. The highly glycosylated S1 domain forms the "head" of the spike protein and contains a receptor binding domain that interacts with 2,3-linked sialic acids on the surface of the host cell (Promkuntod et al. 2014. Virology. 448: 26-32). The S2 domain contains the rest of the extracellular domain (ectodomain) (the "stem"), the transmembrane domain, and the intracellular domain located in the cytoplasm.

[0006] To date, the most widely used live attenuated IBV vaccine strain was developed in the Netherlands in the 1960s by serial passage of a Massachusetts-like IBV strain (Bijlenga et al. 2004; Avian Pathol. 33:550-557). However, since the 1970s, new IBV serotypes have emerged for which traditional Massachusetts-like vaccines do not provide adequate protection (Cook et al. 2012. Avian Pathol. 41:239-250). Therefore, there is still a need for new and highly effective IBV vaccines against other IBV serotypes.

[0007] IBV Beaudette (Geilhausen et al. 1973. Arch Gesamte Virusforsch.: 40(3) (1973), pp. 285-290) and H120 (G. Bijlenga et al. 2004. Avian Pathol.: 33(6); pp. 550-557) are attenuated IBVs. However, attenuation may result in loss of immunogenicity.

[0008] In addition, recombinant IBV has been generated. Zhou et al. 2016 (Arch Virol.; 161:3179-3187) disclosed an H120 (Massachusetts genotype) IBV with a Beaudette (Massachusetts genotype) spike protein. Hodgson et al. 2004 (J Virol 78:13804-13811) disclosed a Beaudette (Massachusetts genotype) IBV with an M41 (Massachusetts genotype) spike protein. In addition, Armesto et al. 2011 (PLoS One: 6(8):e24352) disclosed an IBV Beaudette (Massachusetts genotype) with a heterologous spike protein from 4 / 91 (4 / 91 genotype).

[0009] However, the recombinant IBVs published by Zhou et al. 2016 and Hodgson et al. 2004 cannot be considered as IBVs with heterologous spike proteins, because the IBV and the inserted spike protein are from the same genotype / serotype (Massachusetts). Furthermore, all the vaccines mentioned are based on the Beaudette backbone or have a spike protein from Beaudette.

[0010] Furthermore, despite the fact that Beaudette was described decades ago and recombinant methods using Beaudette have been known for over a decade, no Beaudette-based vaccine or such recombinant vaccine (with a heterologous spike protein) is commercially available. Recombinant IBV based on Beaudette is not suitable as a vaccine. Wei et al 2014 (Apl Microbiol Biotechnol 98) disclosed a Beaudette IBV with the S1 subunit of H120.

[0011] Ellis et al 2018 (J. Virol. 92(23)), Hodgson et al (J. Virol. 78(24)), and Armesto et al 2011 (PLoS One: 6(8): e24352) all disclosed Beaudette IBV with either M41 or 4 / 91 spike proteins. However, Ellis et al 2018 (J. Virol. 92(23)) described recombinant Beaudette with a chimeric spike protein containing heterologous S1 subunits from M41 or QX combined with the S2 subunit of the Beaudette spike protein, which failed to provide adequate protection against homologous challenge with S1 (“Single vaccination of specific pathogen-free chickens with rIBV expressing S1 from virulent strains M41 or QX, i.e., BeauR-M41(S1) and BeauR-QX(S1), provided incomplete protection against homologous challenge based on ciliary motility and clinical signs”; abstract). Furthermore, Ellis et al 2018 (J. Virol. 92 (23)) described that the full-length S gene (S1 and S2 from M41) only provided partial protection against challenge with the homologous serotype IBV (page 12), suggesting that the IBV Beaudette strain is not suitable as a backbone for a recombinant IBV vaccine. Hodgson et al (J. Virol. 78 (24)) further disclosed that the Baudette strain "is also considered to be poorly immunogenic," and therefore, "has never been used as a vaccine strain" (page 13802, left column, second paragraph). Therefore, there is a need to produce new, highly effective IBV vaccines and recombinant IBV vaccines, respectively. In addition, there is a need for highly effective IBV vaccine vectors. Detailed Description of the Invention

[0013] Before describing various aspects of the present invention, it must be noted that, as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural meanings unless the context clearly indicates otherwise. Thus, for example, reference to "an antigen" includes a plurality of 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 those of ordinary skill in the art to which the invention pertains. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, preferred methods, devices, and materials are now described. For the purpose of describing and disclosing cell lines, vectors, and methodologies reported in publications that can be used with the present invention, all publications mentioned herein are incorporated herein by reference in their entirety. Nothing herein should be construed as an admission that the present invention is not entitled to antedate these disclosures by virtue of prior invention.

[0014] Composition of matter

[0015] The present invention solves the problems inherent in the prior art and provides a significant advancement in the art.

[0016] Generally, the present invention provides an H52 IBV (Infectious Bronchitis Virus) encoding a heterologous IBV S (Spike) protein or a fragment thereof.

[0017] The term "H52 IBV" is well known to those skilled in the art. The term "IBV" refers to infectious bronchitis virus. The term "H52" defines a specific strain of IBV. The H52 strain is well known to those skilled in the art and belongs to the Massachusetts genotype. IBV strains are typically distinguished by the coding sequence of the spike protein S1 subunit (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. Serotyping involves treating the virus with neutralizing antibodies.

[0018] Furthermore, H52 and H120 could be distinguished by their higher pathogenicity after administration to young chickens.

[0019] How to obtain H52 IBV is common knowledge among those skilled in the art. H52 IBV strains are commercially available, for example, Nobilis IB H52 (MSD Animal Health), AviPro IB H52 (Lohmann Animal Health GmbH & Co. KG), Bronchovac (Ceva), etc. In addition, McDonald et al. 1980 (Avain Pathology 9:245-259) disclosed that H52 IBV can be obtained through the Central Veterinary Laboratory Rotterdam, Kusters (J. gen Virol 68:343-352) disclosed that H52 IBV can be obtained through the Poultry Health Institute Dorn (now GD Animal Health) in the Netherlands, and Chen et al. 2007 (Avian Pathology 36 (4):269-274) disclosed that H52 IBV can be obtained through the China Institute of Veterinary Drug Control. Furthermore, H52 IBV has been used as a vaccine strain for decades (Bijlenga et al. 2004, Avian Pathology 33(6):550-557) and can therefore be isolated in the field. Methods for isolating and identifying H52 IBV strains are well known to those skilled in the art. For example, H52 IBV strains can be identified as described in Zwaagstra et al. 1992 (J. Clin. Microbiol. 30(1):79-84), Handberg et al. 1999 (Avian Pathology 28:327-335), or Callison et al. 2006 (Journal of Virological Methods 138:60-65). Zwaagstra et al. 1992 and Handberg et al. 1999, for example, disclose Massachusetts-specific primers (for the S and N proteins, respectively) for RT-PCR and sequencing, as well as reference sequences for comparison. In addition, H52 IBV has been sequenced and the genome sequence is available, for example EU 817497. Therefore, the viral genome can be generated by synthesizing its sequence and by applying a reverse genetics system.

[0020] The term "spike protein" refers to a specific protein of IBV that is well known to those skilled in the art. The spike protein is the primary inducer of antibodies and protective immune responses. Furthermore, the spike (S) protein facilitates IBV entry into cells by binding to host cell receptors and mediating viral-cell membrane fusion with the host cell. Furthermore, it determines the tissue and cell tropism of the viral strain.

[0021] The term "heterologous S (spike protein)" means that the spike protein or fragment thereof introduced into H52 IBV is from a different genotype or serotype than H52 IBV. Because H52 is a Massachusetts genotype and serotype, the heterologous spike protein is a non-Massachusetts genotype or serotype.

[0022] The terms "protein," "amino acid," and "polypeptide" are used interchangeably. The term "protein" refers to an amino acid sequence composed of naturally occurring amino acids and their derivatives. Naturally occurring amino acids are well known in the art and are described in standard textbooks of biochemistry. Within an amino acid sequence, the amino acids are linked by peptide bonds. Additionally, the two ends of an amino acid sequence are referred to as the carboxyl terminus (C-terminus) and the amino terminus (N-terminus). The term "protein" includes substantially purified proteins or protein preparations that additionally contain other proteins. Furthermore, the term also refers to protein fragments. Furthermore, it includes chemically modified proteins. Such modifications can be artificial or naturally occurring modifications, such as phosphorylation, glycosylation, myristylation, and the like.

[0023] Furthermore, the present invention also provides an immunogenic composition comprising H52 IBV (infectious bronchitis virus) encoding a heterologous S (spike) protein or a fragment thereof.

[0024] In addition, the present invention also provides an immunogenic composition comprising the IBV (Infectious Bronchitis Virus) described herein. Further, the present invention also provides an immunogenic composition comprising H52 IBV (Infectious Bronchitis Virus) encoding a heterologous S (Spike) protein or a fragment thereof.

[0025] 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. This immune response can 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, more preferably conferring protective immunity against one or more clinical signs of IBV infection. A host is also referred to as a "subject." Preferably, any host or subject described or referred to herein is an avian or poultry species.

[0026] Typically, an "immune response" includes, but is not limited to, the production or activation of one or more of the following: antibodies, B cells, helper T cells, suppressor T cells, and / or cytotoxic T cells and / or gamma-delta T cells specific for one or more antigens contained in the immunogenic compositions of the invention. Preferably, the host will exhibit a protective immune response or a therapeutic response.

[0027] A "protective immune response" or "protective immunity" is evidenced by a reduction or absence of clinical signs normally exhibited by an infected host, a faster recovery time and / or a shortened duration of infection in an infected host, or a reduction in pathogen titers in tissues, body fluids, or excretions.

[0028] An immunogenic composition is described as a "vaccine" if it elicits a protective immune response in the host, whereby resistance to new infection is enhanced and / or the clinical severity of disease is reduced.

[0029] H52-IBV - defined by protein coding sequence

[0030] In another specific aspect of the IBV or immunogenic composition of the invention, the H52 IBV has the nucleotide sequence as shown in EU817497 (SEQ ID NO: 78) or a sequence having at least 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98% or 99.99% sequence identity thereto.

[0031] The term "nucleic acid" or "nucleic acid sequence" or "nucleotide sequence" refers to a polynucleotide including a DNA molecule, an RNA molecule, a cDNA molecule or a derivative thereof. The term encompasses single-stranded and double-stranded polynucleotides. The nucleic acids of the present invention include isolated polynucleotides (i.e., isolated from their natural environment) and genetically modified forms. In addition, chemically modified polynucleotides are also included, including naturally occurring modified polynucleotides, such as glycosylated or methylated polynucleotides, or artificially modified polynucleotides, such as biotinylated polynucleotides. In addition, the terms "nucleic acid" and "polynucleotide" are used interchangeably to refer to any nucleic acid. The terms "nucleic acid" and "polynucleotide" also specifically include nucleic acids composed of bases other than the five biologically occurring bases (adenine, guanine, thymine, cytosine and uracil).

[0032] The term "RNA" refers to any ribonucleic acid. The term encompasses single-stranded and double-stranded RNA. The RNA of the present invention includes isolated RNA (i.e., isolated from its natural environment) and genetically modified forms. In addition, chemically modified RNA is also included, including naturally occurring modified RNA, such as methylated RNA, or artificially modified RNA, such as biotinylated RNA. The term "RNA" also specifically includes RNA composed of bases other than the four biologically occurring nucleotides / bases (adenine, guanine, cytosine and uracil).

[0033] In another specific aspect of the IBV or immunogenic composition of the invention, the H52 IBV strain has a spike (S) protein having the amino acid sequence shown in AF352315 (SEQ ID NO: 79) or a sequence having at least 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98% or 99.99% sequence identity thereto.

[0034] It must be understood that the spike protein or nucleic acid sequence can be used to determine whether any IBV strain is derived from H52. However, since the H52 IBV is used as a backbone and the H52 spike protein or nucleic acid sequence is replaced by a heterologous spike protein or fragment thereof, the final IBV with a heterologous spike protein does not contain any H52 spike protein or only a portion of the H52 spike protein remains.

[0035] In another specific aspect of the IBV or immunogenic composition of the present invention, the H52 IBV strain has a spike (S) protein having the amino acid sequence shown in SEQ ID NO: 1 or a sequence having at least 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98% or 99.99% sequence identity thereto.

[0036] In another specific aspect of the IBV or immunogenic composition of the present invention, the H52 IBV strain has a nucleocapsid (N) protein having an amino acid sequence as shown in AY044185 (SEQ ID NO: 80) or a sequence having at least 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98% or 99.99% sequence identity to at least one of the above sequences.

[0037] In another specific aspect of the IBV or immunogenic composition of the invention, the H52 IBV strain has a nucleocapsid (N) protein having an amino acid sequence as shown in AF352310 (SEQ ID NO: 81) or a sequence having at least 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98% or 99.99% sequence identity thereto.

[0038] In another specific aspect of the IBV or immunogenic composition of the present invention, the H52 IBV strain has a nucleocapsid (N) protein having an amino acid sequence as shown in SEQ ID NO: 2 or a sequence having at least 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98% or 99.99% sequence identity thereto.

[0039] In another specific aspect of the IBV or immunogenic composition of the invention, the H52 IBV strain has an envelope (E) protein having the amino acid sequence shown in AF317210 (SEQ ID NO: 82) or a sequence having at least 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98% or 99.99% sequence identity thereto.

[0040] In another specific aspect of the IBV or immunogenic composition of the present invention, the H52 IBV strain has an envelope (E) protein having the amino acid sequence shown in SEQ ID NO: 3 or a sequence having at least 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98% or 99.99% sequence identity thereto.

[0041] In another specific aspect of the IBV or immunogenic composition of the present invention, the H52 IBV strain has a membrane glycoprotein (M) having the amino acid sequence shown in AF286185 (SEQ ID NO: 83) or a sequence having at least 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98% or 99.99% sequence identity thereto.

[0042] In another specific aspect of the IBV or immunogenic composition of the present invention, the H52 IBV strain has a membrane glycoprotein (M) having an amino acid sequence as shown in SEQ ID NO: 4 or a sequence having at least 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98% or 99.99% sequence identity thereto.

[0043] The terms "identity" or "sequence identity" are known in the art and refer to the relationship between two or more polypeptide sequences or two or more polynucleotide sequences, i.e., the relationship between a reference sequence and a designated sequence to be compared to the reference sequence. Sequence identity is determined by sequence comparison after the designated sequence and the reference sequence have been optimally aligned to produce the highest degree of sequence similarity, and is determined by a match between the sequence strings. Based on this alignment, sequence identity can be determined on a position-by-position basis; for example, if the nucleotide or amino acid residue at a particular position is the same, then the sequences are "identical" at that particular position. The total number of such positional identities is divided by the total number of nucleotides or residues in the reference sequence to provide the percentage of sequence identity. Sequence identity can be readily calculated 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, eds., Humana Press, New Jersey (1994); Sequence Analysis in Molecular Biology, von Heinge, G., Academic Press (1987); Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M. Stockton Press, New York (1991); and Carillo, H., and Lipman, D., SIAM J. Applied Molecular Biology. Math., 48:1073 (1988), which teachings are incorporated herein by reference. Preferred methods for determining sequence identity are designed to give maximum matching between the sequences tested. Methods for determining sequence identity are codified in publicly available computer programs that determine sequence identity between specified sequences.Examples of such programs include, but are not limited to, the GCG program package (Devereux, J., et al., Nucleic Acids Research, 12(1):387 (1984)), BLASTP, BLASTN, and FASTA (Altschul, S. F. et al., J. Molec. Biol., 215:403-410 (1990)). The BLASTX program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S. F. et al., NCVI NLM NIH Bethesda, MD 20894, Altschul, S. F. et al., J. Molec. Biol., 215:403-410 (1990), the teachings of which are incorporated herein by reference). These programs use default gap weights to optimally align sequences to produce the highest level of sequence identity between a specified sequence and a reference sequence. For example, a polynucleotide having a nucleotide sequence that has at least, for example, 85%, preferably 90%, or even more preferably 95% "sequence identity" to a reference nucleotide sequence means that the nucleotide sequence of the specified polynucleotide is identical to the reference sequence, except that the specified polynucleotide sequence may include up to 15, preferably up to 10, or even more preferably up to 5 point mutations per every 100 nucleotides of the reference nucleotide sequence. In other words, in a polynucleotide having a nucleotide sequence that is at least 85%, preferably 90%, or even more preferably 95% identical to a reference nucleotide sequence, up to 15%, preferably 10%, or even more preferably 5% of the nucleotides in the reference nucleotide sequence may be deleted or substituted with another nucleotide, or up to 15%, preferably 10%, or even more preferably 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence. These mutations in the reference sequence may occur at the 5' or 3' terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed between individual nucleotides in the reference sequence, or in one or more contiguous groups within the reference sequence. Similarly, a polypeptide having an amino acid sequence that has at least, for example, 85%, preferably 90%, even more preferably 95% sequence identity to a reference amino acid sequence means that the amino acid sequence of the specified polypeptide is identical to the reference sequence, except that the specified polypeptide sequence may include up to 15, preferably up to 10, even more preferably up to 5 amino acid alterations for every 100 amino acids of the reference amino acid sequence.In other words, to obtain a specified polypeptide sequence having at least 85%, preferably 90%, and even more preferably 95% sequence identity to a reference amino acid sequence, up to 15%, preferably up to 10%, and even more preferably up to 5% of the amino acid residues in the reference sequence can be deleted or substituted with another amino acid, or up to 15%, preferably up to 10%, and even more preferably up to 5% of the total number of amino acids in the reference sequence can be inserted into the reference sequence. These changes in the reference sequence can occur at the amino or carboxyl terminal positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed between individual residues in the reference sequence, or in one or more consecutive groups within the reference sequence. Preferably, conservative amino acid substitutions differ at residue positions that are not identical. However, conservative substitutions are not included as matches when determining sequence identity.

[0044] The terms "identity", "sequence identity" and "percent identity" are used interchangeably herein. For the purposes of the present invention, it is defined herein that in order to determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (for example, a gap can be introduced in the first amino acid sequence or nucleic acid for optimal alignment with the second amino acid or nucleic acid sequence). The amino acid or nucleotide residues at 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 percent identity between the 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) × 100). Preferably, the two sequences have the same length.

[0045] Sequence comparison can be carried out on the full length of the two sequences compared or on the fragment of the two sequences. Usually, the comparison is carried out on the full length of the two sequences compared. However, sequence identity can be compared in the region of, for example, 20, 50, 100 or more continuous amino acid residues.

[0046] The skilled artisan will appreciate that different computer programs can be used to determine the homology between two sequences. For example, a mathematical algorithm can be used to compare sequences and determine the percent identity between two sequences. In a preferred embodiment, the percent identity between two amino acid 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 from http: / / www.accelrys.com / products / gcg / ), using a Blosum 62 matrix or a PAM250 matrix, a gap weight of 16, 14, 12, 10, 8, 6, or 4, and a length weight of 1, 2, 3, 4, 5, or 6. The skilled artisan will appreciate that all of these different parameters will produce slightly different results when different algorithms are used, but the overall percent identity between the two sequences will not change significantly.

[0047] The protein or nucleic acid sequences of the present invention can further be used as a "query sequence" to search public databases, for example, to identify other family members or related sequences. Such searches can be performed using the BLASTN and BLASTP programs (version 2.0), as described in 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. To obtain gapped alignments for comparison purposes, the Gapped BLAST program can be used, as described in Altschul et al. (1997) Nucleic Acids Res. 25(17):3389-3402. When using BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., BLASTP and BLASTN) can be used. See the National Center for Biotechnology Information homepage at http: / / www.ncbi.nlm.nih.gov / .

[0048] As used herein, the term "identical to the sequence of SEQ ID NO: X" is specifically understood to be equivalent to the term "identical to the sequence of SEQ ID NO: X over the length of SEQ ID NO: X" or to the term "identical to the sequence of SEQ ID NO: X over the entire length of SEQ ID NO: X". In this context, "X" is any integer selected from 1-85, and thus "SEQ ID NO: X" represents any SEQ ID NO mentioned herein.

[0049] Heterologous S protein

[0050] IBV strains

[0051] 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. The first IBV serotype identified was the Massachusetts type, but in addition to the initially identified Massachusetts type, several other serotypes have been identified in the United States, including Arkansas and Delaware.

[0052] The IBV strain Beaudette is a Massachusetts-type strain, generated by passage in chicken embryos for at least 150 generations. The IBV strain Beaudette was originally isolated by Beaudette and Hudson (J. Am. Vet. Med. A. 90, 51-60, 1937) and passaged in chicken embryos. Other Massachusetts-type IBV strains besides Beaudette are H120, H52, and M41. The H120 strain has been passaged for 120 generations.

[0053] IBV QX is described as a virulent local isolate of IBV that was originally isolated in China. However, the virus has spread to Europe and has been found in parts of Western Europe, primarily in the Netherlands, but also in Germany, France, Belgium, Denmark, and the United Kingdom. In addition, the QX genotype or serotype has been described in several countries in Asia and Africa.

[0054] IBV 4 / 91, commonly known as 793B, was first reported in the UK in the early 1990s and has now spread to many parts of the world. CR88 is an attenuated strain belonging to this serotype and is commercially available as a vaccine.

[0055] In the late 1990s, strains designated "Italien-02" or "Italy-02" were isolated in Italy. Sequence analysis of one of these isolates was published in 2002 (NCBI-BLAST, accession number AJ457137). However, studies have shown that this Italian-02 strain is widespread in Europe and has become one of the most predominant genotypes in the United Kingdom, Spain, France, and the Netherlands, in addition to IBV variant 4 / 91.

[0056] A new infectious bronchitis virus (IBV) genotype, designated Q1, has been circulating in China since 1996 and was first reported in Italy in 2011. Q1 is associated with increased mortality, renal lesions, and proventriculitis.

[0057] In addition, strains D274, B1648 / D8880, D1466, V1397 and Arkansas have also been identified in Europe.

[0058] How to obtain any IBV strain is common knowledge among those skilled in the art. IBV strains can be purchased commercially, obtained from research institutions, or assembled into complementary DNA, as IBV strains have been sequenced and their sequences published and can therefore be used. In addition, IBV strains can be isolated from the wild. Methods for isolating and identifying IBV strains are well known to those skilled in the art. Valter Leonardo de Quadros 2011 (Dissertation, Das Bronchitis Virus(IBV):Molekularbiologische Untersuchungen zur Diagnostik undzum Vorkommen sowie zur des Genotyps IBV QX in spezifischpathogenfreien(SPF)Broilern,Freie Berlin), Worthington et al. 2009 (Avian Pathology 37(3), 247-257), Liu et al. 2009 (Virus Genes 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) describe how to isolate and differentiate different IBV strains.

[0059] In another specific aspect of the IBV or immunogenic composition of the invention, the heterologous spike protein is of a non-Massachusetts genotype or serotype.

[0060] In another specific aspect of the IBV or immunogenic composition of the invention, the heterologous S protein or fragment thereof is from an IBV having a genotype or serotype selected from the group consisting of 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 97 / 01), and 02), JMK, LDT3, Maine (e.g., Maine 209), 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).

[0061] According to another specific aspect of the IBV or immunogenic composition of the present invention, the heterologous S protein or fragment thereof is from an IBV genotype or serotype selected from the group consisting of: 4 / 91, QX, Q1, Italy 02, Arkansas, Conneticut, Georgia, LDT3, PL84084, variant 2 and Brazil.

[0062] According to another specific aspect of the IBV or immunogenic composition of the present invention, the heterologous S protein or fragment thereof is from an IBV of the following genotypes or serotypes selected from: 4 / 91, QX, Q1, Arkansas, variant 2 and Brazil.

[0063] According to another specific aspect of the IBV or immunogenic composition of the present invention, the 4 / 91 strain is selected from the group consisting of 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, 4 / 91-pathogenic, 4 / 91 attenuated, IB4-91 and CR88.

[0064] According to another specific aspect of the IBV or immunogenic composition of the present invention, the QX strain is selected from the group consisting of 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.

[0065] According to another specific aspect of the IBV or immunogenic composition of the present invention, the Q1 strain is selected from the group consisting of CK / CH / LDL / 98I, CK / CH / LSD / 08-10, J2, Q1, AR08ER22, AR08BA21 and Chile-295-10.

[0066] According to another specific aspect of the IBV or immunogenic composition of the present invention, the Arkansas strain is selected from the group consisting of Ark99, ArkGA, ArkDPI, AL / 5364 / 00, ARKDPI11, AL / 0803 / 01, AL / 7149 / 00, ArkDPI101, AL / 1221 / 01, AL / 1793 / 01 and AL / 4614 / 98.

[0067] According to another specific aspect of the IBV or immunogenic composition of the present invention, the variant 2 strain is selected from the group consisting of 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.

[0068] According to another specific aspect of the IBV or immunogenic composition of the present invention, the Brazil strain is selected from the group consisting of BR-1, BR-2, 23 / 2013 and IBV / Brasil / 351 / 1984.

[0069] According to another specific aspect of the IBV or immunogenic composition of the present invention, the heterologous S protein or fragment thereof is from the 4 / 91 genotype or serotype.

[0070] In another specific aspect of the IBV or immunogenic composition of the invention, the heterologous S protein or fragment thereof is from the 4 / 91 genotype or serotype, which has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98% or 99.99% sequence identity with SEQ ID NO:5 or 6.

[0071] In another specific aspect of the IBV or immunogenic composition of the invention, the heterologous S protein or fragment thereof is from the QX genotype or serotype, which has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98% or 99.99% sequence identity to SEQ ID NO:7 or 8.

[0072] In another specific aspect of the IBV or immunogenic composition of the invention, the heterologous S protein or fragment thereof is from the Q1 genotype or serotype, which has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98% or 99.99% sequence identity with SEQ ID NO: 9 or 10.

[0073] In another specific aspect of the IBV or immunogenic composition of the invention, the heterologous S protein or fragment thereof is from the Arkansas genotype or serotype, which has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98% or 99.99% sequence identity to SEQ ID NO:11 or 12.

[0074] In another specific aspect of the IBV or immunogenic composition of the invention, the heterologous S protein or fragment thereof is from a variant 2 genotype or serotype having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98% or 99.99% sequence identity to SEQ ID NO: 13 or 14.

[0075] In another specific aspect of the IBV or immunogenic composition of the invention, the heterologous S protein or fragment thereof is from the Brazil genotype or serotype, which has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98% or 99.99% sequence identity with SEQ ID NO:15 or 16.

[0076] In another specific aspect of the IBV or immunogenic composition of the invention, the heterologous S protein or fragment thereof is selected from SEQ ID NO: 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16.

[0077] In another specific aspect of the IBV or immunogenic composition of the invention, the heterologous S protein is a full-length spike protein.

[0078] The experimental data of the present invention show that fragments of the spike protein sequence, such as the extracellular domain of the spike protein, can be used. However, the full-length spike protein sequence can also be used.

[0079] In another specific aspect of the IBV or immunogenic composition of the invention, the fragment of the heterologous S (Spike) protein is at least 500, 750, 1000 or 1077 amino acids in length.

[0080] In another specific aspect of the IBV or immunogenic composition of the invention, the fragment of the heterologous S (Spike) protein is at least 500, 750, 1000 or 1077 amino acids in length from the N-terminus.

[0081] The term "N-terminus" is well known to those skilled in the art. The N-terminus is also referred to as the amino terminus, NH2-terminus, N-terminus, or amine terminus. When a protein is translated from messenger RNA, it is produced from the N-terminus to the C-terminus. Thus, the N-terminus is the starting point of the amino acid chain (protein or polypeptide) that contains the amine group (-NH2).

[0082] In another specific aspect of the IBV or immunogenic composition of the invention, the fragment of the heterologous S (Spike) protein is at least 1000 amino acids in length.

[0083] In another specific aspect of the IBV or immunogenic composition of the invention, the fragment of the heterologous S (Spike) protein is the extracellular domain of the Spike protein.

[0084] The term "ectodomain" is well known to those skilled in the art. The spike protein comprises distinct functional parts, namely, a signal sequence, an extracellular domain, a transmembrane domain, and an intracellular domain (from N-terminus to C-terminus). Thus, after cleavage of the signal sequence, the N-terminus of the spike protein begins with the extracellular domain. The length of the IBV spike protein extracellular domain is approximately 1077 amino acids, varying by a few amino acids depending on the IBV strain.

[0085] In another specific aspect of the IBV or immunogenic composition of the invention, the heterologous S (spike) protein or fragment thereof replaces the homologous S protein or fragment thereof.

[0086] In another specific aspect of the IBV or immunogenic composition of the invention, the heterologous S (spike) protein or fragment thereof replaces the naturally occurring S protein or fragment thereof.

[0087] In another specific aspect of the IBV or immunogenic composition of the present invention, the heterologous S (spike) protein or fragment thereof replaces the S protein or fragment thereof in H52 IBV.

[0088] In another specific aspect of the IBV or immunogenic composition of the invention, the IBV is attenuated.

[0089] The term "attenuated" refers to a pathogen that has reduced virulence compared to a wild-type isolate. In the present invention, an attenuated IBV is a strain in which virulence has been reduced so that it does not cause clinical signs of IBV infection but is able to induce an immune response in a target animal, but can also refer to a reduction in the occurrence or severity of clinical signs in an animal infected with attenuated IBV compared to a "control" animal that is not infected with attenuated IBV or does not receive attenuated virus. In this context, the term "reduced / reduced" refers to a reduction of at least 10%, preferably 25%, even more preferably 50%, even 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 attenuated IBV as defined above. Therefore, an attenuated IBV strain is a strain suitable for incorporation into an immunogenic composition comprising a modified live IBV.

[0090] In another specific aspect of the IBV or immunogenic composition of the invention, the IBV is inactivated.

[0091] Any conventional inactivation method can be used for the purpose of the present invention. Therefore, inactivation can be carried out by chemical and / or physical treatment methods known to those skilled in the art. Preferred inactivation methods include adding cyclized binary ethyleneimine (BEI), including adding 2-bromoethyleneamine hydrobromide (BEA) solution that has been cyclized to binary ethyleneimine (BEI). Preferred other chemical inactivators include but are not limited to Triton X-100, sodium deoxycholate, hexadecyltrimethylammonium bromide, β-propiolactone, thimerosal, phenol and formaldehyde (formalin). However, inactivation can also include a neutralization step. Preferred neutralizing agents include but are not limited to sodium thiosulfate, sodium bisulfite, etc.

[0092] Preferred formalin inactivation conditions include a formalin concentration of about 0.02% (v / v) to 2.0% (v / v), more preferably about 0.1% (v / v) to 1.0% (v / v), still more preferably about 0.15% (v / v) to 0.8% (v / v), even more preferably about 0.16% (v / v) to 0.6% (v / v), and most preferably about 0.2% (v / v) to 0.4% (v / v). The incubation time depends on the resistance of the IBV. Generally, the inactivation procedure is performed until no IBV growth is detected in a suitable culture system.

[0093] Preferably, the inactivated IBV of the present invention is formalin-inactivated, preferably using the concentrations described herein above.

[0094] The inactivated IBV of the present invention can be incorporated into liposomes using known techniques, such as described in Nature 1974, 252, 252-254 or Journal of Immunology, 1978, 120, 1109-13. In another embodiment of the present invention, the inactivated IBV of the present invention can be conjugated to a suitable biological compound such as a polysaccharide, peptide, protein, or the like, or a combination thereof.

[0095] In another specific aspect of the IBV or immunogenic composition of the invention, the IBV is genetically engineered.

[0096] The term "genetically engineered" refers to an IBV that has been mutated using a "reverse genetics" approach. Preferably, the IBV of the present invention has been genetically engineered. Reverse genetics techniques involve the preparation of synthetic recombinant viral RNA. However, "reverse genetics" techniques are well known to those skilled in the art.

[0097] In another specific aspect of the IBV or immunogenic composition of the invention, the IBV is a recombinant IBV.

[0098] As used herein, the term "recombinant" refers to an RNA genome (or RNA sequence, cDNA sequence or protein) that has any modification that is not naturally present 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 to be "recombinant" if it contains insertions, deletions, inversions, relocations or point mutations that are artificially introduced, for example, by human intervention. Thus, the RNA genome sequence (or RNA sequence, cDNA sequence or protein) is not associated with all or part of the sequence (or RNA sequence, cDNA sequence or protein) that is naturally associated with it. The term "recombinant" used with respect to viruses refers to viruses produced by artificial manipulation of the viral genome. The term "recombinant virus" includes genetically modified viruses.

[0099] In another specific aspect of the IBV or immunogenic composition of the invention, the IBV is chimeric.

[0100] The term "chimeric" refers to an IBV that contains one or more nucleotide sequences from another coronavirus, preferably from another IBV strain. For example, IBV H52 encoding a heterologous S (spike) protein or a fragment thereof is a chimeric IBV.

[0101] In another specific aspect of the immunogenic composition according to the present invention, the immunogenic composition is a vaccine. The term "vaccine" has been described elsewhere herein. However, if the host exhibits a protective immune response, whereby resistance to new infection is increased and / or the clinical severity of the disease is reduced, the immunogenic composition is described as a "vaccine."

[0102] In another specific aspect of the immunogenic composition according to the present invention, the immunogenic composition comprises a pharmaceutically acceptable carrier.

[0103] The term "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, stabilizers, diluents, preservatives, antibacterial and antifungal agents, isotonicity agents, adsorption delaying agents, adjuvants, immunostimulants, and combinations thereof.

[0104] "Diluents" may include water, saline, glucose, ethanol, glycerol, etc. Isotonic agents may include sodium chloride, glucose, mannitol, sorbitol, lactose, etc. Stabilizers include albumin and alkali metal salts of ethylenediaminetetraacetic acid, etc.

[0105] In another specific aspect of the immunogenic composition of the present invention, the pharmaceutically acceptable carrier is phosphate buffered saline.

[0106] Preferably, the immunogenic composition further comprises a sucrose gelatin stabilizer.

[0107] Preferably, the pharmaceutically acceptable carrier is chitosan.

[0108] Chitosan is a natural deacetylated polysaccharide derived from chitin in crustaceans (such as shrimp and crabs), insects, and other invertebrates. Recently, Rauw et al. (2009) (Vet Immunol Immunop 134:249-258) demonstrated that chitosan enhanced the cellular immune response of a live Newcastle disease vaccine, promoting its protective effect. Furthermore, Wang et al. (2012) (Arch Virol (2012) 157:1451-1461) demonstrated the potential of chitosan as an adjuvant for attenuated live influenza vaccines.

[0109] Preferably, the immunogenic composition may further include one or more other immunomodulators, such as interleukins, interferons or other cytokines.The amounts and concentrations of adjuvants and additives useful within the scope of the present invention can be readily determined by a skilled artisan.

[0110] In certain aspects, the immunogenic compositions of the present invention contain an adjuvant. As used herein, "adjuvants" may include aluminum hydroxide and aluminum phosphate, saponins such as Quil A, QS-21 (Cambridge Biotech, Cambridge, Massachusetts), GPI-0100 (Pharmaceuticals, Inc., Birmingham, Alabama), water-in-oil emulsions, oil-in-water emulsions, and water-in-oil-in-water emulsions. The emulsions may be based, in particular, on light liquid paraffin oil (European typical); isoprenoid oils such as squalane or squalene; oils produced from oligomers of alkenes, particularly isobutylene or decene. Esters of acids or alcohols containing straight-chain alkyl groups, particularly vegetable oils, ethyl oleate, propylene glycol di(caprylate / caprate), glyceryl tri(caprylate / caprate), or propylene glycol dioleate. Esters of branched fatty acids or alcohols, particularly isostearates. The oil may be used in combination with an emulsifier to form an emulsion. Emulsifiers are preferably nonionic surfactants, in particular sorbitol, mannitol (e.g., anhydromannitol oleate), ethylene glycol, polyglycerol, propylene glycol, and esters of oleic, isostearic, ricinoleic, or hydroxystearic acids, which may be optionally ethoxylated, and polyoxypropylene-polyoxyethylene copolymer blocks, in particular Pluronic products, particularly L121. See Hunter et al., Theory and Practice of Adjuvants (Ed. Stewart-Toole, DES), John Wiley & Sons, Agricultural Industry Standard, pp. 51-94 (1995) and Todd et al., Vaccine 15: 564-570 (1997). Typical adjuvants are the SPT emulsions described in Vaccine Design, Subunits, and Adjuvant Methods, ed. M. Powell and M. Newman, Plenum Press, 1995, p. 147, and the MF59 emulsions described in the same book, p. 183.

[0111] Another example of an adjuvant is a compound selected from a polymer of acrylic acid or methacrylic acid and a copolymer of maleic anhydride and an alkenyl derivative. Advantageous adjuvant compounds are cross-linked polymers of acrylic acid or methacrylic acid, particularly polymers cross-linked with polyalkenyl ethers of sugars or polyols. These compounds are known as carbomers (Phameuropa Volume 8, Issue 2, June 1996). Those skilled in the art may also refer to U.S. Patent No. 2,909,462, which describes such acrylic acid polymers cross-linked with polyols having at least 3 hydroxyl groups, preferably no more than 8 hydroxyl groups, wherein the hydrogen atoms of at least 3 hydroxyl groups are substituted with unsaturated aliphatic groups having at least 2 carbon atoms. Preferred groups are those containing 2 to 4 carbon atoms, such as vinyl, allyl, and other vinyl unsaturated groups. The unsaturated group itself may contain other substituents, such as methyl. Products sold under the name Carbopol (BFGoodrich, Ohio, USA) are particularly suitable. They are cross-linked with allyl sucrose or allyl pentaerythritol. Among them, Carbopol 974P, 934P and 971P can be mentioned. The most commonly used is Carbopol 971P. Among the copolymers of maleic anhydride and alkenyl derivatives, there is the copolymer EMA (Monsanto Company), which is a copolymer of maleic anhydride and ethylene. These polymers are dissolved in water to produce an acidic solution, which is then neutralized, preferably to physiological pH, in order to prepare an adjuvant solution to be incorporated into an immunogenic, immunological or vaccine composition.

[0112] Further suitable adjuvants include, but are not limited to, the RIBI adjuvant system (RIBI, Inc.), Block copolymers (CytRx, Atlanta, Georgia), SAF-M (Chiron, Emeryville, California), monophosphoryl lipid A, Avridine lipid amine adjuvant, heat-labile endotoxin from Escherichia coli (recombinant or otherwise), cholera toxin, IMS1314 or murine amido dipeptide, or naturally occurring or recombinant cytokines or analogs thereof, or stimulators of endogenous cytokine release, and the like.

[0113] It is expected that the adjuvant will 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. Furthermore, the adjuvant concentration can be about 0.01 to 50% by volume of the final product, preferably about 2% to 30%, more preferably about 5% to 25%, more preferably about 7% to 22%, and most preferably a concentration of 10% to 20%.

[0114] In another specific aspect of the immunogenic composition according to the present invention, the immunogenic composition is effective in treating and / or preventing clinical signs caused by IBV in a subject in need thereof. The terms "treating and / or preventing", "clinical symptoms" and "in need thereof" have been defined elsewhere.

[0115] In another specific aspect of the immunogenic composition according to the present invention, the immunogenic composition protects against challenge with an IBV strain having a genotype or serotype of a heterologous spike protein.

[0116] In another specific aspect of the immunogenic composition according to the present invention, the immunogenic composition protects against challenge with strains of the 4 / 91, QX, Q1, Arkansas, variant 2 or Brazil genotype.

[0117] In another specific aspect of the immunogenic composition according to the present invention, the immunogenic composition protects against challenge with a 4 / 91 genotype strain.

[0118] In another specific aspect of the immunogenic composition according to the present invention, the immunogenic composition is formulated for single-dose administration.

[0119] The volume of a single dose has been defined elsewhere herein.

[0120] Furthermore, it has been shown that one dose of the immunological composition of the present invention is effective following administration of such a single dose of the immunological composition of the present invention.

[0121] In another specific aspect of the immunogenic composition according to the invention, the immunogenic composition is administered subcutaneously, intramuscularly, orally, in ovo, by spray, by drinking water or by eye drops.

[0122] In another specific aspect of the immunogenic composition according to the present invention, the immunogenic composition comprises 1 to 10 log 10 EID 50 of the IBV.

[0123] In another specific aspect of the immunogenic composition according to the present invention, the immunogenic composition comprises 2 to 5 log 10 EID 50 of the IBV.

[0124] In another specific aspect of the immunogenic composition according to the present invention, the immunogenic composition comprises 2 to 4 log 10 EID 50 of the IBV.

[0125] Reagent test kit

[0126] If desired, the composition may be present in a package or dispensing device containing one or more unit dosage forms comprising the active ingredient. For example, the package may comprise metal or plastic foil, such as a blister pack. The package or dispenser device may be accompanied by instructions for administration, preferably for administration to a subject, particularly poultry. Associated with such containers may be a notice in the form of regulations of a governmental agency governing the manufacture, use, or sale of pharmaceuticals or biological products, which notice reflects the agency's permission for the preparation, use, or sale of the drug for human use.

[0127] Thus, the present invention provides a kit comprising an IBV or immunogenic composition as described herein.

[0128] In a specific aspect of the kit according to the present invention, the kit further comprises instructions for use in treating and / or preventing poultry diseases.

[0129] In a specific aspect of the kit according to the present invention, the kit further comprises instructions for treating and / or preventing poultry diseases.

[0130] In a particular aspect of the kit according to the present invention, the kit further comprises instructions for use in the treatment and / or prevention of IB (infectious bronchitis).

[0131] Treatment

[0132] Furthermore, the present invention provides methods for immunizing a subject comprising administering to the subject an immunogenic composition as described herein.

[0133] The term "immunization" refers to active immunization by administering an immunogenic composition to a subject to be immunized, thereby eliciting an immune response against the antigen contained in such immunogenic composition.

[0134] Preferably, immunization results in a reduced incidence of a specific IBV infection in a population or a reduced severity of clinical signs caused by or associated with a specific IBV infection.

[0135] In addition, immunization of a subject in need thereof with the immunogenic composition provided herein results in the subject being protected from infection with IBV. Even more ideally, the immunization results in an effective, long-lasting immune response to IBV infection. It will be understood that the time period will last for more than 1 month, preferably more than 2 months, preferably more than 3 months, more preferably more than 4 months, more preferably more than 5 months, and more preferably more than 6 months. It should be understood that immunization is not necessarily effective for all immunized subjects. However, the term requires that a substantial portion of the subjects in the population be effectively immunized.

[0136] Preferably, in this context, a population of subjects is considered that would normally develop clinical signs typically caused by or associated with IBV infection, i.e., unimmunized. Whether such subjects have been effectively immunized can be further determined by one skilled in the art. Preferably, immunization is effective if 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 population have a reduction in the incidence or severity of clinical signs of 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%, more preferably at least 80%, more preferably at least 90%, more preferably at least 95%, and most preferably 100%, compared to unimmunized subjects or subjects immunized with a previously available immunogenic composition of the present invention who are subsequently infected with a specific IBV.

[0137] Furthermore, the present invention provides a method of treating or preventing clinical signs caused by IBV in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an immunogenic composition as described herein.

[0138] The term "treating or preventing" refers to reducing the incidence of a specific IBV infection in a population, or reducing the severity of clinical signs caused by or associated with a specific IBV infection. Therefore, the term "treating or preventing" also refers to reducing the number of subjects infected with a specific IBV in a population (= reducing the incidence of a specific IBV infection), or reducing the severity of clinical signs typically associated with or caused by an IBV infection, or reducing viral shedding following infection with a specific IBV, or preventing or reducing egg drop in laying hens following infection with a specific IBV, in comparison to a group of subjects that have not received such an immunogenic composition.

[0139] "Treatment or prevention" generally involves administering an effective amount of an immunogenic composition of the present invention to a subject or group of subjects in need of or who would benefit from such treatment / prevention. The term "treatment" refers to administering an effective amount of an immunogenic composition once a subject or at least some of the subjects in the group have been infected with such IBV and wherein such subjects have shown some clinical signs caused by or associated with such IBV infection. The term "prevention" refers to administering to a subject before the subject has been infected with IBV, or at least when no subject in the subject or group of subjects has shown any clinical signs caused by or associated with such IBV infection. The terms "prevention" and "prevent" are used interchangeably in this application.

[0140] As used herein, the term "effective amount" refers to, but is not limited to, an amount of an antigen that elicits or is capable of eliciting an immune response in a subject. Such an effective amount can reduce the incidence of a specific IBV infection in a population or reduce the severity of clinical signs of a specific IBV infection.

[0141] Preferably, the incidence or severity of clinical symptoms is reduced 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%, more preferably at least 80%, more preferably at least 90%, more preferably at least 95%, and most preferably 100%, compared to a subject who has not received treatment or who has received the immunogenic composition available before the present invention but who subsequently becomes infected with the specific IBV.

[0142] As used herein, the term "clinical signs" refers to signs that a subject is infected with IBV. The clinical signs of infection depend on the pathogen selected. Examples of such clinical signs include, but are not limited to, respiratory distress, nephritis, salphingitis, abnormal egg production, ruffled feathers, depression, decreased growth rate, and decreased appetite. Signs of respiratory distress include respiratory signs, including wheezing, coughing, sneezing, tracheal rales, nasal and ocular discharge, tracheal lesions, and ciliostasis within the trachea. Signs of nephritis include kidney lesions and watery diarrhea. Signs of abnormal egg production include reduced egg production, smaller egg size, poorer eggshells, decreased internal egg quality, thinner egg whites, and ciliostasis within the oviducts. However, clinical signs also include, but are not limited to, clinical signs that can be observed directly in live animals. Examples of clinical signs that can be observed directly in live animals include nasal and ocular discharge, coughing, wheezing, sneezing, tracheal rales, ruffled feathers, conjunctivitis, weight loss, decreased growth rate, decreased appetite, dehydration, watery diarrhea, lameness, lethargy, emaciation, and inactivity.

[0143] Preferably, the reduction in the incidence or severity of clinical symptoms in a treated subject refers to a reduction in ciliary arrest, a reduction in rales, a reduction in egg dropping, a reduction in renal lesions, a reduction in watery diarrhea, a reduction in weight loss, a reduction in viral load, a reduction in viral shedding, or a combination thereof, compared to a subject who has not received treatment or who has received a previously available immunogenic composition of the present invention but who is subsequently infected with a particular IBV.

[0144] As used herein, the term "need" or "in need thereof" means that the administration / treatment is associated with enhancing or improving health or clinical signs or having any other positive medical effect on the health of the subject receiving the immunogenic composition of the present invention.

[0145] The terms "reduce" or "reduction" or "alleviate" or "lower" are used interchangeably herein. The term "reduce" refers to a reduction in clinical signs of 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%, more preferably at least 80%, more preferably at least 90%, more preferably at least 95%, and most preferably 100%, compared to a subject who has not received treatment (not immunized) but is subsequently infected with a particular IBV.

[0146] Additionally, the present invention provides methods of reducing ciliary arrest in a subject in need thereof, compared to a non-immunized control group of subjects of the same species, comprising administering to the subject a therapeutically effective amount of an immunogenic composition as described herein.

[0147] As shown in the Examples, the immunogenic compositions provided herein have been demonstrated to be effective in reducing ciliary arrest.

[0148] The term "ciliostasis" refers to a decrease in the movement of cilia in the trachea. Therefore, ciliary arrest can be determined by examining the lining of the tracheal rings to determine the movement of cilia. How to determine the movement of cilia in the trachea is within the general knowledge of those skilled in the art.

[0149] Preferably, ciliary motility is not reduced starting from day 10 after challenge or infection, more preferably starting from day 5 after challenge or infection, more preferably starting from day 4 after challenge or infection, more preferably starting from day 3 after challenge or infection, and most preferably starting from day 1 or day 2 after challenge or infection with IBV, compared to subjects in a non-immune control group of the same species.

[0150] The term "reduction of ciliary arrest" refers to a reduction of ciliary arrest 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%, compared to a non-immunized control group of the same species. How to measure a reduction in ciliary arrest is within the general knowledge of those skilled in the art.

[0151] In one aspect of the invention, the subject is an avian.

[0152] The term "avian" is well known to those skilled in the art. The term "avian" includes all birds, including poultry.

[0153] In one aspect of the invention, the subject is poultry.

[0154] 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. Furthermore, the term "chicken" includes broilers, laying hens, and the breeding stock of both, also known as breeders.

[0155] In one aspect of the invention, the subject is selected from the group consisting of chicken, turkey, quail or pheasant.

[0156] In one aspect of the invention, the subject is a chicken.

[0157] In one aspect of the invention, the immunogenic composition is administered once.

[0158] A single dose is understood to be administered only once. As shown in the Examples, the immunogenic compositions provided herein have been demonstrated to have therapeutic efficacy following administration of a single dose to a subject in need thereof.

[0159] The dose volume per bird depends on the route of administration and the age of the bird.

[0160] Typically, at any age, eye drop vaccines are administered in a volume of 1 to 100 μl per dose. Preferably, the total volume of a single dose of the eye drop vaccine is between about 5 μl and 70 μl, more preferably between about 20 μl and 50 μl, preferably a single dose of 20 μl, 25 μl, 30 μl, 35 μl, 40 μl, 45 μl or 50 μl. Preferably, the total volume of a single dose of the eye drop vaccine is between about 5 μl and 70 μl, more preferably between about 20 μl and 50 μl, preferably a single dose of 20 μl, 25 μl, 30 μl, 35 μl, 40 μl, 45 μl or 50 μl.

[0161] For day-old poultry, the spray vaccine may comprise a dose volume of 25 to 1000 μl. Preferably, the total volume of a single dose of the spray vaccine is between about 50 μl and 5000 μl, more preferably between about 75 μl and 2000 μl, more preferably between about 100 μl and 1000 μl, even more preferably between about 200 μl and 900 μl, more preferably between about 300 μl and 800 μl, even more preferably between about 400 μl and 700 μl, preferably a single dose 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. Most preferably the total volume of a single dose is 400 μl, 450 μl, 500 μl, 550 μl, 600 μl, 650 μl or 700 μl.

[0162] Vaccines for in ovo vaccination may comprise a dose volume of 50 to 100 μl, preferably 50 μl. Preferably, the total volume of a single dose of the in ovo vaccine is from about 10 μl to 250 μl, more preferably from about 15 μl to 200 μl, even more preferably from about 20 μl to 150 μl, even more preferably from about 30 μl to 100 μl, even more preferably from about 30 μl to 75 μl, and preferably a single dose of 30 μl, 35 μl, 40 μl, 45 μl, 50 μl, 55 μl, 60 μl, 65 μl, 70 μl, or 75 μl. Most preferably, the total volume of a single dose is 40 μl, 45 μl, 50 μl, 55 μl, or 60 μl.

[0163] A vaccine for intramuscular or subcutaneous administration or a single dose of drinking water vaccine may contain a volume of 30 μl to 1000 μl. Preferably, the total volume of a single dose is between about 30 μl and 1000 μl, more preferably between about 50 μl and 500 μl, more preferably between about 75 μl and 250 μl, even more preferably between about 100 μl and 200 μl, and most preferably a single dose 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.

[0164] In one aspect of the invention, the immunogenic composition is administered in two or more doses.

[0165] However, the immunogenic composition may be administered in two or more doses, with a first dose being administered before a second dose (boost dose).

[0166] In preferred aspects 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 regimens, alternative embodiments also include other 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, and the time frame between each dose is consistent with the time frame between the first and second doses described above.

[0167] Preferably, the first administration of the vaccine is carried out within three weeks of age, more preferably within one week of age, and most preferably by the following method at one day of age. The second administration can be carried out within 20 weeks of age, preferably within 16-18 weeks of age, more preferably within 6-12 weeks of age. Exemplarily, an initial (first) vaccination is carried out at 1-10 days of age, and a second vaccination (enhancement) is carried out using a live vaccine or an inactivated vaccine at 6-12 or 16-18 weeks of age. More preferably, an initial (first) vaccination is carried out at one day of age, and a second vaccination (enhancement) is carried out using a live vaccine or an inactivated vaccine at 6-12 or 16-18 weeks of age.

[0168] Where an in ovo vaccine is used, the first administration is preferably given when the embryo is between 15 and 19 days old, preferably on day 17, 18 or 19, and most preferably on day 18. The second administration may be given within the first three weeks of life, and preferably within the first 10 days of life.

[0169] In one aspect of the invention, the immunogenic composition is administered subcutaneously, intramuscularly, orally, in ovo, via spray, via drinking water or via eye drops.

[0170] The immunogenic composition is preferably administered topically or systemically. Suitable routes of administration for conventional use are oral or parenteral administration, such as intranasal, intravenous, intradermal, transdermal, intramuscular, intraperitoneal, subcutaneous, and inhalation, in ovo, by spraying, by drinking water, or by eye drops. However, depending on the properties and mode of action of the compound, the immunogenic composition may also be administered by other routes. For example, such other routes include transdermal, intravenous, intravascular, intraarterial, intranasal, intrathoracic, intratracheal, intracardiac, intraperitoneal, intraperitoneal, intramedullary, extraperitoneal, intrarectal, and intravaginal. However, most preferably, the immunogenic composition is administered subcutaneously, intramuscularly, orally, in ovo, by spraying, by drinking water, or by eye drops.

[0171] The live IBV vaccine is preferably administered alone via eye drops, intranasally, intramuscularly or subcutaneously.

[0172] More preferably, a number of administration methods are used, including drinking water and aerosol vaccination.Also preferred is the use of the vaccine as an embryonic vaccine (so called in ovo vaccine), as further described below.

[0173] For example, broilers can be vaccinated at one day old or 1-3 weeks of age, especially for broilers with high MDA levels. Layers or breeders can initially be vaccinated at 1-10 days of age and boosted with the vaccine at 7-12 or 16-18 weeks of age.

[0174] In ovo administration

[0175] As described above, the present invention also provides an IBV vaccine that can be safely administered via the intra-ovo route while simultaneously inducing a protective immune response. In-ovo administration is well known to those skilled in the art, and those skilled in the art can perform intra-ovo administration without much effort. In-ovo administration of the vaccine involves administering the vaccine to an avian embryo contained in an egg (for a review of intra-ovo vaccination, see: Ricks et al., Veterinary Advances. 495-515, 1999). The vaccine can be injected into any suitable compartment of the egg (such as the allantoic fluid, yolk sac, amniotic membrane, air cell, or into the embryo), as described in the art (Sharma; Am. J. Vet. Res. 45 1619-1623, 1984). Preferably, the vaccine is administered below the shell (air cell) membrane and the chorioallantoic membrane.

[0176] Preferably, the vaccine is injected into embryonic eggs at a later stage of embryonic development, typically in the last quarter of the incubation period, preferably 3-4 days before hatching. This is preferably done when the embryos are 15 to 19 days old, preferably on days 17, 18, or 19, and ideally on day 18. The vaccinated embryonic eggs are then transferred to an incubator for incubation. The in ovo administration process can be automated using robotic injection techniques described in the prior art.

[0177] Conventional vaccines typically used for post-hatch vaccination of poultry cannot be used for in ovo vaccination because late embryos are highly sensitive to most vaccine viruses examined. However, International Patent Application WO 01 / 64244 discloses that IBV vaccines can be administered in ovo, but only at very low doses. Furthermore, Wakenell et al., 1986 (Am. J. Vet. Res., 47933-938) disclose that passage of IB vaccine viruses in tissue culture renders them nonpathogenic to embryos.

[0178] In one aspect of the invention, the immunogenic composition is administered by eye drops.

[0179] Typically, live vaccines for post-hatch administration consist of attenuated IBV at a concentration of 10 1 to 10 8 EID 50 (50% egg infectious dose), preferably 10 per dose 2 to 10 5 EID 50 , more preferably 10 per dose 2 to 10 4 EID 50 , even more preferably 10 per dose 2 to 10 3 EID 50 .

[0180] Live vaccines for in ovo administration typically contain 10 2 -10 7 EID 50 / embryo, preferably 10 2 -10 3 EID 50 / embryo-attenuated IBV.

[0181] Preferably, the immunogenic compositions of the present invention include the IBV of the present invention in an amount of about 1 to about 10 log 10 EID (Egg Infectious Dose) 50 , preferably from about 2 to about 8 log per dose 10 EID 50 , preferably from about 2 to about 7 log per dose 10 EID 50 , more preferably from about 2 to about 6 log per dose 10 EID 50 Even more preferably, about 2 to about 5 log per dose 10 EID 50 Even more preferably, about 2 to about 4 log per dose 10 EID 50 The amount of , most preferably about 2 to about 3 log per dose 10 EID 50 More preferably, the immunogenic compositions of the invention include the IBV of the invention in 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 1, 2, 3, 4, 5, 6, 7, 8 ... 10 EID 50 .

[0182] In one aspect of the invention, the immunogenic composition comprises 1 to 10 log 10 IBV of EID50.

[0183] In one aspect of the invention, the immunogenic composition comprises 2 to 5 log 10 IBV of EID50.

[0184] In one aspect of the invention, the immunogenic composition comprises 2-4 log 10 IBV of EID50.

[0185] In one aspect of the invention, the immunogenic composition is administered to a subject within one week of age, within three days of age, within two days of age, or within one day of age.

[0186] Preferably, the subject to be immunized is 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 subject to be immunized is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days old. Most preferably, the subject to be immunized is 1, 2, 3, 4, 5, 6, or 7 days old.

[0187] However, it must be understood that it does take several days for the poultry's immune system to build up immunity to IBV infection after vaccination of the subject. Therefore, vaccination is best performed within the first 24 hours of the subject's life.

[0188] In one aspect of the invention, the immunogenic composition is administered to a subject within the first day of life.As shown in the Examples, the immunogenic compositions provided herein have been shown to be safe and effective when administered to one-day-old poultry.

[0189] In one aspect of the invention, the method results in an improvement in an efficacy parameter selected from the group consisting of prevention or reduction of ciliary arrest, prevention or reduction of rales, prevention or reduction of egg loss, prevention or reduction of renal lesions, prevention or reduction of watery diarrhea, reduction in weight loss, reduction in viral load, reduction in viral shedding, or a combination thereof, compared to subjects in an untreated control group of the same species.

[0190] The terms "treating and / or preventing" have been defined elsewhere, wherein the terms "preventing" and "preventing" or "avoiding" are used interchangeably in this application. In addition, the term "shedding" has also been defined elsewhere.

[0191] The terms "reduce", "lower", "alleviate" or "become lower" refer to a decrease in efficacy parameters (ciliostasis, rales, egg loss, kidney lesions, watery diarrhea, weight loss, viral load, viral shedding) 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% compared to a non-immunized control group of the same species. How to measure the improvement of efficacy parameters is common knowledge to those skilled in the art.

[0192] The term "viral load" is well known to those skilled in the art. The term viral load is used interchangeably with viral titer herein. Viral load or viral titer is a measure of the severity of an active viral infection and can be determined by methods known to those skilled in the art. Determination can be based on the detection of viral proteins, such as by antibodies binding to viral proteins and further detecting them, or by detecting viral RNA by amplification methods such as RT-PCR. Monitoring viral RNA associated with the virus in plasma by nucleic acid amplification methods is a widely used parameter for assessing the status and progression of retroviral diseases and for assessing the effectiveness of preventive and therapeutic interventions. For example, viral load or viral titer can be calculated by estimating the number of viable viruses in the body fluid in question, such as the number of RNA copies per milliliter of plasma.

[0193] The term "ciliostasis" is well known to those skilled in the art. The surface of the trachea is covered with specialized epithelial cells lined with numerous, mobile, hair-like structures called cilia. The term "ciliostasis" encompasses a reduction or loss of cilia and / or a loss or partial loss of ciliary activity. Ciliosis can be determined without undue effort by those skilled in the art.

[0194] The term "rales" is well known to those skilled in the art. However, the term "rales" includes tracheal rales and refers to sounds emitted from the bronchi. Those skilled in the art can determine rales without undue effort.

[0195] The term "egg drop" is well known to those skilled in the art. The term "egg drop" includes a reduction in egg production.

[0196] In one aspect of the invention, treatment or prevention results in prevention or reduction of ciliary arrest compared to an untreated control group of subjects of the same species.

[0197] In one aspect of the invention, the treatment or prevention results in the prevention or reduction of kidney pathology compared to an untreated control group of subjects of the same species.

[0198] In one aspect of the invention, the treatment or prevention results in the prevention or reduction of egg drop compared to an untreated control group of subjects of the same species.

[0199] The present invention further provides an IBV or immunogenic composition as described herein for use in therapeutic treatment.

[0200] The present invention further provides an IBV or immunogenic composition as described herein for use as an immunogen or vaccine.

[0201] The present invention also provides an IBV or immunogenic composition as described herein for use as a medicament.

[0202] The present invention further provides use of the IBV or immunogenic composition described herein in the preparation of a medicament.

[0203] The present invention further provides use of an IBV or immunogenic composition described herein for treating and / or preventing an IBV infection in a subject.

[0204] The present invention further provides an immunogenic composition comprising an H52 IBV (infectious bronchitis virus) encoding a heterologous S (spike) protein or a fragment thereof, wherein the H52 IBV comprises a nucleocapsid (N) protein, an envelope (E) protein or a membrane glycoprotein (M) having a sequence such as SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, AY044185 (SEQ ID NO 80), AF352310 (SEQ ID NO 81), AF317210 (SEQ ID NO 82) or AF286185 (SEQ ID NO 83). NO:83) or a sequence having at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98% or 99.99% sequence identity thereto, and wherein the heterologous S protein or fragment thereof is selected from the genotype or serotype of 4 / 91, QX, Q1, Arkansas, variant 2 and Brazil, or is selected from the genotype or serotype of SEQ ID NO:83) or a sequence having at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98% or 99.99% sequence identity thereto. The amino acid sequence shown in NO:5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98% or 99.99% sequence identity thereto.

[0205] In another specific aspect of the immunogenic composition according to the present invention, the heterologous S protein is a full-length spike protein.

[0206] In another specific aspect of the immunogenic composition according to the invention, the fragment of the heterologous S (spike) protein has a length of at least 500, 750, 1000 or 1077 amino acids starting from the N-terminus.

[0207] In another specific aspect of the immunogenic composition according to the present invention, the fragment of the heterologous S (Spike) protein is the extracellular domain of the Spike protein.

[0208] In another specific aspect of the immunogenic composition according to the invention, the IBV is attenuated.

[0209] The present invention further provides a method for preparing an immunogenic composition for treating and / or preventing IBV infection in a subject, the method comprising:

[0210] a.) providing an H52 IBV comprising a spike (S) protein, a nucleocapsid (N) protein, an envelope (E) protein, or a membrane glycoprotein (M) having an amino acid sequence as set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, AF352315 (SEQ ID NO 79), AY044185 (SEQ ID NO 80), AF352310 (SEQ ID NO 81), AF317210 (SEQ ID NO 82), or AF286185 (SEQ ID NO: 83), or a sequence having at least 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98%, or 99.99% sequence identity thereto; and

[0211] b.) providing a heterologous S protein or fragment thereof selected from the list of genotypes or serotypes of 4 / 91, QX, Q1, Arkansas, variant 2, and Brazil, or selected from the amino acid sequence of SEQ ID NO: 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98%, or 99.99% sequence identity thereto; and

[0212] c.) replacing the spike protein or fragment thereof of H52 IBV in a) with the heterologous S (spike) protein or fragment thereof described in b) to obtain H52 IBV having a heterologous S protein or fragment thereof; and

[0213] d.) obtaining the H52 IBV having a heterologous S protein or a fragment thereof; and

[0214] e.) Adding a pharmaceutically acceptable carrier.

[0215] The term "obtaining" includes harvesting, separation, purification and / or formulation (eg, completion, inactivation and / or mixing) of said IBV H52 with a heterologous S protein or a fragment thereof.

[0216] The term "harvesting" refers to collecting or recovering the IBV H52 containing the heterologous S protein or fragment thereof from transfected or infected cells or cell lines. Any conventional method known in the art can be used, such as any separation method. Well-known methods in the art include centrifugation or filtration, for example, using a semipermeable membrane with a certain pore size.

[0217] The term "isolation" includes the step of isolating the IBV H52 containing the heterologous S protein or fragment thereof. Methods for isolating the IBV H52 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 cycling, sonication, and the like.

[0218] Methods for "purifying" the IBV H52 containing the heterologous S protein or fragment thereof from an isolate are known to those skilled in the art, for example, as described in Methods of Protein Purification: A Practical Approach (E.L.V. Harris and S. Angel, eds., 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 chelate chromatography, ion exchange chromatography, covalent chromatography, hydrophobic interaction chromatography, and the like. The vector can be obtained in a purified form, or be free or substantially free of other cellular material or culture medium, etc. Following separation and / or purification, the antigen exhibits a purity of at least 80%, preferably 80%-90%, more preferably 90%-97%, and most preferably greater than 97%, up to an absolutely pure form free of any contaminants.

[0219] According to another aspect, "obtaining" as used herein may also include further finishing steps as part of the final formulation process, such as addition of buffers, inactivation, neutralization steps, etc.

[0220] In another specific aspect of the method for preparing an immunogenic composition according to the present invention, the fragment of the heterologous S (Spike) protein is the extracellular domain of the Spike protein.

[0221] In another specific aspect of the immunogenic composition according to the present invention, the pharmaceutically acceptable carrier is selected from solvents, dispersion media, coatings, stabilizers, diluents, preservatives, antibacterial and antifungal agents, isotonic agents, adsorption delaying agents, adjuvants, immunostimulants and combinations thereof.

[0222] In another specific aspect of the method for preparing an immunogenic composition according to the present invention, the heterologous S protein is a full-length spike protein.

[0223] The present invention further relates to a plasmid comprising a nucleic acid encoding a portion of the H52 IBV (Infectious Bronchitis Virus) genome, including a heterologous IBV S (Spike) protein or a fragment thereof, such as the pUC57-s H52 rIBVCR88 S Ecto donor plasmid (SEQ ID NO: 21).

[0224] Terms

[0225] This article also describes the following terms:

[0226] 1. H52 IBV (Infectious Bronchitis Virus) encoding a heterologous IBV S (Spike) protein or a fragment thereof.

[0227] 2. An immunogenic composition comprising H52 IBV (Infectious Bronchitis Virus) encoding a heterologous S (Spike) protein or a fragment thereof.

[0228] 3. An immunogenic composition comprising IBV (Infectious Bronchitis Virus) according to clause 1.

[0229] IBV H52 - Protein coding sequence definition

[0230] 4. The IBV or immunogenic composition of any one of clauses 1 to 3, wherein the H52 IBV has, consists of or comprises the nucleotide sequence shown in EU817497 (SEQ ID NO 78), or a sequence having at least 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98% or 99.99% sequence identity thereto.

[0231] 5. The IBV or immunogenic composition of any one of clauses 1 to 4, wherein the H52 IBV has, consists of or comprises a Spike (S1) protein having the amino acid sequence shown in AF352315 (SEQ ID NO 79) or a sequence having at least 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98% or 99.99% sequence identity thereto.

[0232] 6. The immunogenic composition of IBV or any one of clauses 1 to 5, wherein the H52 IBV strain has, consists of, or comprises a spike (S) protein having the amino acid sequence of SEQ ID NO 1 or a sequence having at least 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98% or 99.99% sequence identity thereto.

[0233] 7. The IBV or immunogenic composition of any one of clauses 1 to 6, wherein the H52 IBV has, consists of or comprises a nucleocapsid (N) protein having the amino acid sequence of AY044185 (SEQ ID NO 80) or AF352310 (SEQ ID NO 81 ), or a sequence having at least 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98% or 99.99% sequence identity thereto.

[0234] 8. The IBV or immunogenic composition of any one of clauses 1 to 7, wherein the H52 IBV has, consists of, or comprises a nucleocapsid (N) protein having the amino acid sequence shown in SEQ ID NO: 2 or a sequence having at least 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98% or 99.99% sequence identity thereto.

[0235] 9. The IBV or immunogenic composition of any one of clauses 1 to 8, wherein the H52 IBV has, consists of or comprises an envelope (E) protein having the amino acid sequence of AF317210 (SEQ ID NO 82) or a sequence having at least 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98% or 99.99% sequence identity thereto.

[0236] 10. The IBV or immunogenic composition of any one of clauses 1 to 9, wherein the H52 IBV has, consists of or comprises an envelope (E) protein having the amino acid sequence of SEQ ID NO: 3 or a sequence having at least 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98% or 99.99% sequence identity thereto.

[0237] 11. The IBV or immunogenic composition of any one of clauses 1 to 10, wherein the H52 IBV has, consists of or comprises a membrane glycoprotein (M) having the amino acid sequence shown as AF286185 (SEQ ID NO 83) or a sequence having at least 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98% or 99.99% sequence identity thereto.

[0238] 12. The IBV or immunogenic composition of any one of clauses 1 to 11, wherein the H52 IBV has, consists of, or comprises a membrane glycoprotein (M) protein having the amino acid sequence of SEQ ID NO: 4, or a sequence having at least 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98% or 99.99% sequence identity thereto.

[0239] Heterologous S protein

[0240] 13. The IBV or immunogenic composition of any one of clauses 1 to 12, wherein the heterologous spike is a non-Massachusetts genotype or serotype.

[0241] 14. The IBV or immunogenic composition of any one of clauses 1 to 13, wherein the heterologous S protein or fragment thereof is from an IBV having a genotype or serotype selected from the group consisting of 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 (e.g., Italy 02), JMK, LDT3, Maine (e.g., Maine 209), 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).

[0242] 15. The IBV or immunogenic composition of any one of clauses 1 to 14, wherein the heterologous S protein or fragment thereof is from an IBV genotype or serotype selected from the group consisting of: 4 / 91, QX, Q1, Italy 02, Arkansas, Conneticut, Georgia, LDT3, PL84084, variant 2 or Brazil.

[0243] 16. The IBV or immunogenic composition of any one of clauses 1 to 15, wherein the heterologous S protein or fragment thereof is from an IBV of a genotype or serotype selected from the group consisting of: 4 / 91, QX, Q1, Arkansas, variant 2, and Brazil.

[0244] 17. The IBV or immunogenic composition of clause 16, wherein the 4 / 91 strain is selected from the group consisting of: 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, 4 / 91-pathogenic, 4 / 91-attenuated, IB4-91 and CR88.

[0245] 18. The IBV or immunogenic composition of clause 16, wherein the QX strain is selected from the group consisting of: 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.

[0246] 19. The IBV or immunogenic composition of clause 16, wherein the Q1 strain is selected from the group consisting of: CK / CH / LDL / 98I, CK / CH / LSD / 08-10, J2, Q1, AR08ER22, AR08BA21 and Chile-295-10.

[0247] 20. The IBV or immunogenic composition of clause 16, wherein the Arkansas strain is selected from the group consisting of: Ark99, ArkGA, ArkDPI, AL / 5364 / 00, ARKDPI11, AL / 0803 / 01, AL / 7149 / 00, ArkDPI101, AL / 1221 / 01, AL / 1793 / 01 and AL / 4614 / 98.

[0248] 21. The IBV or immunogenic composition of clause 16, wherein the variant 2 strain is selected from the group consisting of: 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.

[0249] 22. The IBV or immunogenic composition of clause 16, wherein the Brazil strain is selected from BR-1, BR-2, 23 / 2013 and IBV / Brasil / 351 / 1984.

[0250] 23. The IBV or immunogenic composition of any one of clauses 1 to 22, wherein the heterologous S protein or fragment thereof is from the 4 / 91 genotype or serotype.

[0251] 24. The IBV or immunogenic composition of any one of clauses 1 to 23, wherein the heterologous S protein or fragment thereof is from a 4 / 91 genotype or serotype having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98% or 99.99% sequence identity to SEQ ID NO: 5 or 6, or the heterologous S protein or fragment thereof comprises or consists of SEQ ID NO: NO:5 or 6, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98% or 99.99% sequence identity thereto.

[0252] 25. The IBV or immunogenic composition of any one of clauses 1 to 24, wherein the heterologous S protein or fragment thereof is from a QX genotype or serotype having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98% or 99.99% sequence identity to SEQ ID NO: 7 or 8, or the heterologous S protein or fragment thereof comprises or consists of SEQ ID NO: 8. The invention also comprises the amino acid sequence of NO:7 or 8, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98% or 99.99% sequence identity thereto.

[0253] 26. The IBV or immunogenic composition of any one of clauses 1 to 25, wherein the heterologous S protein or fragment thereof is from a Q1 genotype or serotype having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98% or 99.99% sequence identity to SEQ ID NO: 9 or 10, or the heterologous S protein or fragment thereof comprises or consists of SEQ ID NO: The invention also comprises the amino acid sequence of NO:9 or 10, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98% or 99.99% sequence identity thereto.

[0254] 27. The IBV or immunogenic composition of any one of clauses 1 to 26, wherein the heterologous S protein or fragment thereof is from an Arkansas genotype or serotype having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98% or 99.99% sequence identity to SEQ ID NO: 11 or 12, or the heterologous S protein or fragment thereof comprises or consists of SEQ ID NO: The amino acid sequence of NO:11 or 12, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98% or 99.99% sequence identity thereto.

[0255] 28. The IBV or immunogenic composition of any one of clauses 1 to 27, wherein the heterologous S protein or fragment thereof is from a variant 2 genotype or serotype having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98% or 99.99% sequence identity to SEQ ID NO: 13 or 14, or the heterologous S protein or fragment thereof comprises or consists of SEQ ID NO: The invention also comprises the amino acid sequence of NO:13 or 14, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98% or 99.99% sequence identity thereto.

[0256] 29. The IBV or immunogenic composition of any one of clauses 1 to 28, wherein the heterologous S protein or fragment thereof is from a Brazil genotype or serotype having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98% or 99.99% sequence identity to SEQ ID NO: 15 or 16, or the heterologous S protein or fragment thereof comprises or consists of SEQ ID NO: The invention also comprises the amino acid sequence of NO:15 or 16, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98% or 99.99% sequence identity thereto.

[0257] 30. The IBV or immunogenic composition of any one of clauses 1 to 29, wherein the heterologous S protein or fragment thereof is selected from SEQ ID NO: 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16.

[0258] 31. The IBV or immunogenic composition of any one of clauses 1 to 30, wherein the heterologous S protein is a full-length spike protein.

[0259] 32. The IBV or immunogenic composition of any one of clauses 1 to 31 , wherein the fragment of the heterologous S (Spike) protein is at least 500, 750, 1000 or 1077 amino acids in length.

[0260] 33. The IBV or immunogenic composition of any one of clauses 1 to 32, wherein the fragment of the heterologous S (Spike) protein is at least 500, 750, 1000 or 1077 amino acids in length from the N-terminus.

[0261] 34. The IBV or immunogenic composition of any one of clauses 1 to 33, wherein the fragment of the heterologous S (Spike) protein is at least 1000 amino acids in length.

[0262] 35. The IBV or immunogenic composition of any one of clauses 1 to 34, wherein the heterologous S (Spike) protein is the extracellular domain of the Spike protein.

[0263] 36. The IBV or immunogenic composition of any one of clauses 1 to 35, wherein the heterologous S (Spike) protein or fragment thereof replaces the homologous S protein or fragment thereof.

[0264] 37. The IBV or immunogenic composition of any one of clauses 1 to 36, wherein the heterologous S (Spike) protein or fragment thereof replaces a naturally occurring S protein or fragment thereof.

[0265] 38. The IBV or immunogenic composition of any one of clauses 1 to 37, wherein the heterologous S (Spike) protein or fragment thereof replaces the S protein or fragment thereof of H52.

[0266] 39. The IBV or immunogenic composition of any one of clauses 1 to 38, wherein the IBV is attenuated.

[0267] 40. The IBV or immunogenic composition of any one of clauses 1 to 39, wherein the IBV is inactivated.

[0268] 41. The IBV or immunogenic composition of any one of clauses 1 to 40, wherein the IBV is genetically engineered.

[0269] 42. The IBV or immunogenic composition of any one of clauses 1 to 41 wherein the IBV is a recombinant IBV.

[0270] 43. The immunogenic composition of any one of clauses 2 to 42 wherein the immunogenic composition is a vaccine.

[0271] 44. The immunogenic composition of any one of clauses 2 to 43, wherein the immunogenic composition comprises a pharmaceutically acceptable carrier.

[0272] 45. The immunogenic composition of clause 44, wherein the pharmaceutically acceptable carrier is phosphate buffered saline.

[0273] 46. ​​The immunogenic composition of any one of clauses 2 to 45, wherein the immunogenic composition is effective in treating and / or preventing clinical signs caused by IBV in a subject in need thereof.

[0274] 47. The immunogenic composition of any one of clauses 2 to 46, wherein the immunogenic composition protects against challenge with an IBV strain having a serotype with a heterologous spike protein.

[0275] 48. The immunogenic composition of any one of clauses 2 to 47 wherein the immunogenic composition protects against challenge with a 4 / 91, QX, Q1, Arkansas, variant 2 or Brazil genotype strain.

[0276] 49. The immunogenic composition of any one of clauses 2 to 48, wherein the immunogenic composition protects against challenge with a genotype 4 / 91 strain.

[0277] 50. The immunogenic composition of any one of clauses 2 to 49 wherein the immunogenic composition is formulated for single dose administration.

[0278] 51. The immunogenic composition of any one of clauses 2 to 50 wherein the immunogenic composition is administered subcutaneously, intramuscularly, orally, in ovo, by spray, by drinking water or as eye drops.

[0279] 52. The immunogenic composition of any one of clauses 2 to 51, wherein the immunogenic composition comprises 1 to 10 log 10 EID 50 / dose of IBV.

[0280] 53. The immunogenic composition of any one of clauses 2 to 52, wherein the immunogenic composition comprises 2 to 5 log 10 EID 50 / dose of IBV.

[0281] 54. The immunogenic composition of any one of clauses 2 to 53, wherein the immunogenic composition comprises 2 to 4 log 10 EID 50 / dose of IBV.

[0282] 55. A kit comprising the IBV or immunogenic composition of any one of clauses 1-54.

[0283] 56. The kit according to clause 55, wherein the kit further comprises instructions for use in treating and / or preventing avian diseases.

[0284] 57. The kit according to clause 55, wherein the kit further comprises instructions for use in treating and / or preventing poultry diseases.

[0285] 58. A kit according to clause 55, wherein the kit further comprises instructions for use in treating and / or preventing IB.

[0286] 59. A method of immunizing a subject comprising administering to the subject an immunogenic composition according to any one of clauses 2 to 54.

[0287] 60. A method of treating or preventing clinical signs caused by IBV in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the immunogenic composition of any one of clauses 2 to 54.

[0288] 61. A method of reducing ciliary arrest in a subject in need thereof 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 according to any one of clauses 2-54.

[0289] 62. The immunogenic composition of any one of clauses 2 to 54 for use in a method of immunizing a subject, the method comprising administering to the subject a therapeutically effective amount of the immunogenic composition.

[0290] 63. The immunogenic composition of any one of clauses 2 to 54 for use in a method of treating or preventing clinical signs caused by IBV in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the immunogenic composition.

[0291] 64. The immunogenic composition of any one of clauses 2 to 54 for use in a method of reducing ciliary arrest in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the immunogenic composition.

[0292] 65. The method or use of any one of clauses 59 to 64, wherein the subject is a poultry.

[0293] 66. The method or use of any one of clauses 59 to 65, wherein the subject is poultry.

[0294] 67. The method or use of any one of clauses 59 to 66, wherein the subject is selected from a chicken, a turkey, a quail or a pheasant.

[0295] 68. The method or use of any one of clauses 59 to 67, wherein the subject is a chicken.

[0296] 69. The method or use of any one of clauses 59 to 68 wherein the immunogenic composition is administered once.

[0297] 70. The method or use of any one of clauses 59 to 69 wherein the immunogenic composition is administered in two or more doses.

[0298] 71. The method or use of any one of clauses 59 to 70 wherein the immunogenic composition is administered subcutaneously, intramuscularly, orally, by in ovo injection, by spray, by drinking water or as eye drops.

[0299] 72. The method or use of any one of clauses 59 to 71 wherein the immunogenic composition is administered by eye drops.

[0300] 73. The method or use of any one of clauses 59 to 72, wherein the immunogenic composition comprises 1 to 10 log 10 EID 50 / dose of IBV.

[0301] 74. The method or use of any one of clauses 59 to 73, wherein the immunogenic composition comprises 2 to 5 log 10 EID 50 / dose of IBV.

[0302] 75. The method or use of any one of clauses 59 to 74, wherein the immunogenic composition comprises 2 to 4 log 10 EID 50 / dose of IBV.

[0303] 76. The method or use of any one of clauses 59 to 75, wherein the immunogenic composition is administered to the subject within the first week, within the first three days, within the first two days, or within the first day of the subject's age.

[0304] 77. The method or use of any one of clauses 59 to 76, wherein the immunogenic composition is administered to the subject within the first day of the subject's age.

[0305] 78. The method or use of any one of clauses 59 to 77, wherein the method results in an improvement in an efficacy parameter selected from the group consisting of prevention or reduction of ciliary arrest, prevention or reduction of rales, prevention or reduction of egg loss, prevention or reduction of renal lesions, prevention or reduction of watery diarrhea, prevention or reduction of weight loss, reduction of viral load, reduction of viral shedding, or a combination thereof, compared to an untreated control subject of the same species.

[0306] 79. The method or use of any one of clauses 59 to 78, wherein the treating or preventing results in prevention or reduction of ciliary arrest compared to an untreated control subject of the same species.

[0307] 80. The method or use of any one of clauses 59 to 79, wherein the treating or preventing results in the prevention or reduction of kidney pathology compared to an untreated control subject of the same species.

[0308] 81. The method or use of any one of clauses 59 to 80, wherein the treatment or prevention results in the prevention or reduction of egg loss compared to an untreated control group of subjects of the same species.

[0309] 82. The IBV or immunogenic composition of any one of clauses 1 to 54 for therapeutic use.

[0310] 83. The IBV or immunogenic composition of any one of clauses 1 to 54 for use as an immunogen or vaccine.

[0311] 84. The IBV or immunogenic composition of any one of clauses 1 to 54 for use as a medicament.

[0312] 85. Use of the IBV or immunogenic composition of any one of clauses 1 to 54 in the manufacture of a medicament.

[0313] 86. Use of the IBV or immunogenic composition of any one of clauses 1 to 54 for treating and / or preventing IBV infection in a subject.

[0314] 87. An immunogenic composition comprising an H52 IBV (infectious bronchitis virus) encoding a heterologous S (spike) protein or a fragment thereof, wherein the H52 IBV comprises a nucleocapsid (N) protein, an envelope (E) protein or a membrane glycoprotein (M) having or comprising or consisting of the sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, AY044185 (SEQ ID NO 80), AF352310 (SEQ ID NO 81), AF317210 (SEQ ID NO 82) or AF286185 (SEQ ID NO: 83). ID NO: 83) or a sequence having at least 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98% or 99.99% sequence identity thereto, and wherein the heterologous S protein or fragment thereof is selected from the following genotypes or serotypes: 4, 91, QX, Q1, Arkansas, variant 2 and Brazil, or is selected from the following amino acid sequences: SEQ ID NO: 83) or a sequence having at least 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98% or 99.99% sequence identity thereto, and wherein the heterologous S protein or fragment thereof is selected from the following genotypes or serotypes: 4, 91, QX, Q1, Arkansas, variant 2 and Brazil, or is selected from the following amino acid sequences: NO:5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98% or 99.99% sequence identity thereto, or a heterologous S protein or fragment thereof comprising or consisting of the following amino acid sequence: SEQ ID NO:5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98% or 99.99% sequence identity thereto NO: 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98% or 99.99% sequence identity thereto.

[0315] 88. The immunogenic composition of clause 87, wherein the heterologous S protein is a full-length spike protein.

[0316] 89. The immunogenic composition of clause 87, wherein the fragment of the heterologous S (spike) protein is at least 500, 750, 1000 or 1077 amino acids in length from the N-terminus.

[0317] 90. The immunogenic composition of clause 87 or 89, wherein the fragment of the heterologous S (Spike) protein is the extracellular domain of the Spike protein.

[0318] 91. The immunogenic composition of any one of clauses 92 to 95 wherein the IBV is attenuated.

[0319] 92. A method of preparing an immunogenic composition for treating and / or preventing IBV infection in a subject, comprising:

[0320] a.) providing an H52 IBV comprising a spike (S) protein, a nucleocapsid (N) protein, an envelope (E) protein, or a membrane glycoprotein (M) having, comprising, or consisting of the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, AF352315 (SEQ ID NO 79), AY044185 (SEQ ID NO 80), AF352310 (SEQ ID NO 81), AF317210 (SEQ ID NO 82), or AF286185 (SEQ ID NO: 83), or a sequence having at least 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98%, or 99.99% sequence identity thereto; and

[0321] b.) providing a heterologous S protein or a fragment thereof selected from the genotype or serotype of 4 / 91, QX, Q1, Arkansas, variant 2 and Brazil, or selected from the amino acid sequence of SEQ ID NO: 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98% or 99.99% sequence identity thereto, or providing a heterologous S protein or a fragment thereof comprising or consisting of SEQ ID NO: NO:5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98% or 99.99% sequence identity thereto; and

[0322] c.) replacing the spike protein of H52 IBV of a) with the heterologous S (spike) protein or a fragment thereof of b) so that H52 IBV has a heterologous S protein or a fragment thereof; and

[0323] d.) obtaining the H52 IBV having a heterologous S protein or a fragment thereof; and

[0324] e.) Adding a pharmaceutically acceptable carrier.

[0325] 93. The method of clause 92, wherein the fragment of the heterologous S (spike) protein is the extracellular domain of the spike protein.

[0326] 94. The method of clause 92 or 93, wherein the pharmaceutically acceptable carrier is selected from the group consisting of solvents, dispersion media, coating agents, stabilizers, diluents, preservatives, antibacterial and antifungal agents, isotonicity agents, adsorption delaying agents, adjuvants, immunostimulants, and combinations thereof.

[0327] 95. The method of any one of clauses 92 or 94, wherein the heterologous S protein is a full-length spike protein. BRIEF DESCRIPTION OF THE DRAWINGS

[0329] Figure 1 In ovo replication kinetics of H52 rIBV CR88 Secto compared to recombinant wild-type viruses CR88 and H52. Data points represent the mean of five samples per time point. Error bars represent standard deviation.

[0330] Figure 2 Overview of Ciliary Arrest Scoring The sum of the 10 individual scores for the 10 rings of an animal was calculated and represented by a single point in the graph. Maximum ciliary arrest corresponds to a score of 40, while no ciliary arrest corresponds to a score of 0. Means and significance (p<0.0007) were calculated using GraphPad Prism and a conventional one-way ANOVA test.

[0331] Figure 3 Summary of RT-qPCR results for kidney tissue. Each bird is represented as one data point.

[0332] Figure 4 Summary of nasal swab eluate RT-PCR results. Each individual bird is represented as one data point.

[0333] Figure 5 Overview of Ciliary Arrest Scoring The sum of the 10 individual scores for the 10 rings of an animal was calculated and represented by a single point in the graph. Maximum ciliary arrest corresponds to a score of 40, while no ciliary arrest corresponds to a score of 0. Means and significance (p<0.0001) were calculated using GraphPad Prism and a conventional one-way ANOVA test.

[0334] Sequence Overview

[0335] SEQ ID NO: 1: H52 IBV spike (S) protein.

[0336] SEQ ID NO: 2: H52 IBV nucleocapsid (N) protein.

[0337] SEQ ID NO: 3: H52 IBV envelope (E) protein.

[0338] SEQ ID NO: 4: H52 IBV membrane glycoprotein (M).

[0339] SEQ ID NO: 5 and 6: Heterologous S protein or fragments thereof from 4 / 91 genotype or serotype.

[0340] SEQ ID NO: 7 and 8: Heterologous S protein from QX genotype or serotype or fragments thereof.

[0341] SEQ ID NO: 9 and 10: Heterologous S protein from Q1 genotype or serotype or fragments thereof.

[0342] SEQ ID NOs: 11 and 12: Heterologous S protein from the Arkansas genotype or serotype or fragments thereof.

[0343] SEQ ID NO: 13 and 14: Heterologous S protein or fragment thereof from variant 2 genotype or serotype.

[0344] SEQ ID NO: 15 and 16: Heterologous S protein from Brazil genotype or serotype or fragments thereof.

[0345] SEQ ID NO: 17: IBV CR88 spike protein nucleic acid coding sequence.

[0346] SEQ ID NO: 18: IBV H52 spike protein extracellular domain nucleic acid coding sequence.

[0347] SEQ ID NO: 19: pUC57-s H52 rIBV donor plasmid.

[0348] SEQ ID NO: 20: IBV CR88 spike protein extracellular domain nucleic acid coding sequence.

[0349] SEQ ID NO: 21: pUC57-s H52 rIBV CR88 S Ecto donor plasmid.

[0350] SEQ ID NO: 22 to SEQ ID NO: 77: Primers.

[0351] SEQ ID NO: 78: EU817497 (H52 IBV nucleotide sequence)

[0352] SEQ ID NO:79: AF352315 (H52 IBV S protein amino acid sequence)

[0353] SEQ ID NO:80:AY044185 (H52 IBV N protein amino acid sequence)

[0354] SEQ ID NO:81: AF352310 (H52 IBV N protein amino acid sequence)

[0355] SEQ ID NO:82: AF317210 (H52 IBV E protein amino acid sequence)

[0356] SEQ ID NO:83:AF286185 (H52 IBV M protein amino acid sequence)

[0357] SEQ ID NO: 84 and 85: Primers Example

[0358] The following examples are provided to illustrate specific embodiments of the present invention. These examples are merely examples and should not be construed as limiting the scope or underlying principles of the present invention.

[0359] Example 1: Generation of recombinant IBV H52, wherein the coding sequence of the H52 spike protein or spike protein ectodomain is replaced by the coding sequence of a heterologous spike protein or spike protein ectodomain

[0360] Construction of donor plasmid

[0361] An example of replacing the H52 spike protein ectodomain with the CR88 ectodomain is described in detail: The IBV CR88 spike protein nucleic acid coding sequence (SEQ ID NO: 17) was synthesized by a commercial supplier. It was used as a template to replace the IBV H52 spike protein ectodomain nucleic acid coding sequence (SEQ ID NO: 18) in the pUC57-s IBV-5-1b-S-SIR-3T donor plasmid described by van Beurden et al. (Virol J. 2017; 14(1): 109), hereinafter referred to as the pUC57-s H52 rIBV donor plasmid (SEQ ID NO: 19). Bases 1717 to 4941 of SEQ ID NO: 19 were replaced with the corresponding IBV CR88 spike protein ectodomain nucleic acid coding sequence (SEQ ID NO: 20), which correspond to bases 55 to 3285 of SEQ ID NO 15, respectively. This generated the pUC57-s H52 rIBV CR88 S Ecto donor plasmid (SEQ ID NO: 21), in which the IBV CR88 spike protein ectodomain is encoded from bases 1717 to 4947. To this end, the pUC57-s H52 rIBV donor plasmid (SEQ ID NO: 19) was digested with unique restriction enzyme sites 5' (EcoRV) and 3' (PmlI) close to the H52 spike protein coding sequence to linearize the plasmid and remove the H52 spike protein and flanking sequences. The QIAquick gel extraction kit (Qiagen) was used to purify the band corresponding to the pUC57-sIBV H52 backbone without the H52 spike protein coding sequence. The CR88 spike protein ectodomain coding sequence and flanking 5' and 3' IBV H52 sequences were amplified in three separate PCR reactions using high-fidelity DNA polymerase (NEB; see Table 1 for primers). PCR products were purified by QIAquick gel extraction (Qiagen) and used for Gibson assembly with the HiBuild DNA Assembly Cloning Kit (NEB) according to the kit protocol to generate the pUC57-s H52 rIBV CR88S Ecto donor plasmid.

[0362] Table 1. Gibson assembly primers designed by NEB using the NEBuilder online tool and used to generate PCR products for assembling the pUC57-s H52 rIBV CR88 S Ecto donor plasmid.

[0363]

[0364] Targeted RNA recombination and rescue of recombinant IBV

[0365] For the generation of recombinant IBV, the method of targeted RNA recombination as described by van Beurden et al. (Virol J. 2017; 14(1): 109) was used. Briefly, H52 murinized (m)IBV was generated as described. To generate H52 rIBVCR88 S Ecto, LR7 cells were infected with H52 mIBV and electroporated with in vitro transcripts generated from the pUC57-s H52 rIBV CR88 S Ecto donor plasmid, which were then injected into 8-day-old embryonated SPF chicken eggs (VALOBioMedia). After incubation for 9 days at 37.5°C and 60% humidity, the cells were cloned and expressed using the QIAamp Viral RNAmini Kit (Qiagen) and a Platinum PCR kit. TM SuperScript Taq DNA polymerase (ThermoFisher) TM After RNA was isolated using the III one-step RT-PCR system, allantoic fluid from all eggs was analyzed for rescued recombinant IBV. Primers PO1323 and PO1729 (Table 2) that bind to the H52 IBV 1ab and CR88 IBV S extracellular domains were used, which are specific for recombinant IBV but not for mIBV. ​​The allantoic fluid from the egg inoculated with the highest dilution of LR7 cells was selected for two rounds of endpoint dilution in 8-day-old SPF chicken eggs. The MagMAX™ Core Nucleic Acid Purification Kit (ThermoFisher) and the KingFisher DNA Purification Kit (KingFisher) were used. TM Nucleic acid isolation of limiting dilution samples was performed using the Duo Prime purification system (ThermoFisher), and subsequent analysis of the presence of rIBV was performed by RT-PCR as described above. After the second limiting dilution, positive allantoic fluid from eggs inoculated at the highest dilution was used to propagate in 10-day-old embryonated SPF eggs. The allantoic fluid was diluted 1:1000 in 1xPBS, and 100 μl was injected into each egg. The allantoic fluid was harvested 48 hours after inoculation, cleared of debris, and stored at -80°C. To confirm the sequence of the generated rIBV from the donor plasmid, viral nucleic acid was isolated using the QIAamp viral RNA mini kit, followed by SuperScript III one-step RT-PCR using the primers listed in Table 3. QIAquick PCR purification and subsequent Sanger sequencing using the same primers were performed by commercial suppliers.

[0366] Table 2 Primers for identifying recombinant IBV after rescue

[0367]

[0368] Table 3 Sequencing primers for confirming the donor region sequence in H52 rIBV CR88 S Ecto

[0369]

[0370] Generation and characterization of H52 recombinant IBV in which the coding sequence of the H52 spike protein or spike protein ectodomain is replaced by the coding sequence of the spike protein or spike protein ectodomain of another IBV genotype

[0371] The same methods as those described for producing H52 rIBV CR88 S Ecto were applied to produce and characterize recombinant H52 IBV, wherein the spike protein coding sequence (bases 1663 to 5151 of SEQ ID NO: 19) or the H52 spike protein extracellular domain coding sequence (bases 1717 to 4941 of SEQ ID NO: 19) was replaced with the coding sequence of the IBV spike protein or spike protein extracellular domain of the serotype and genotype listed in Table 4.

[0372] Table 4 Primers used for Gibson assembly of H52 rIBV donor plasmids with heterologous spike protein or spike protein ectodomain

[0373]

[0374]

[0375]

[0376] The primers in Tables 2 and 3 were used to identify and sequence different recombinant viruses and were modified to target the corresponding spike protein sequences when necessary.

[0377] Example 2: In ovo replication dynamics

[0378] Eight eight-day-old embryonated chicken eggs were inoculated with 10 2 EID 50of rIBV and respective controls. The eggs were incubated at 37.5°C and 60% humidity and candidized daily after 0, 8, 24, 34, 48 and 72 hours of incubation, and embryonic mortality was recorded. Five pre-selected eggs were taken for each sample and time point and transferred to 4°C for at least 2 hours. Subsequently, the allantoic fluid was collected and stored at -80°C. For analysis, the samples were thawed and diluted 1:10 in 1xPBS without Ca and Mg, nucleic acids were extracted using the QIAamp DNA Blood Mini kit (Qiagen), and carrier RNA was added using a Hamilton Starlet automatic pipette. The relative amount of IBV RNA in the extracted nucleic acids was analyzed by RT-qPCR, using the protocol of Callison et al. (J Virol Methods. 2006; 138(1-2): 60-5) with some modifications. In brief, the same primers and probes were used, and the temperature protocol was adjusted to suit the use Fast Virus 1-Step Master Mix (ThermoFisher) and ABI TM 7900HT Fast Real-Time PCR System (Thermo Fisher Scientific). All nucleic acid samples were analyzed in triplicate, using 10-fold serial dilutions of IBV H52 as a reference.

[0379] Compared to the recombinant wild-type viruses H52 and CR88, H52 rIBV CR88 S Ecto observed slightly similar replication kinetics at early time points. However, after 32 hours, all viruses reached comparable ct values. All embryos were alive 32 hours after inoculation, while all remaining embryos in all samples died at the 48-hour time point after infection. Therefore, H52 rIBV CR88 S Ecto replicates as efficiently as wild-type virus (see Figure 1 ).

[0380] Example 3: Preparation of vaccines and challenge viruses

[0381] To demonstrate the efficacy of H52 rIBV with heterologous spike protein or spike protein ectodomain in chickens, an aliquot of the virus stock was thawed, diluted 10-fold in 1x PBS, and the 50% embryo infectious dose (EID) was determined by inoculating 100 μl of each dilution into five 8-day-old embryonated chicken eggs. 50). The eggs were incubated at 37.5°C and 60% humidity until 7 days after inoculation. Eggs with dead embryos after 24 hours were excluded from the experiment. All other eggs with dead embryos 7 days after inoculation were considered positive. On the 7th day after inoculation, all eggs with live embryos were candidized from the bottom to identify the bantam chicks considered positive. The EID was calculated using the formula of Reed and Muench 50 / ml (Am J Epidemiol, 1938; 27(3): 493-497). For immunization, the virus stock solution was diluted in 1xPBS to obtain 10 4.3 EID 50 / ml titer (10 per chicken in 50 μl) 3 EID 50 ).

[0382] Challenge viruses of genotypes and serotypes 793B, QX, Q1, Ark, variant 2, and Brazil were propagated in 10-day-old embryonated SPF chicken eggs. 24 hours after inoculation, the eggs were transferred to 4°C for at least 2 hours. The allantoic fluid was collected, aliquoted, and stored at -80°C. Viral titers were determined as described above. The titer was set to 10 by diluting with 1x PBS. 4.3 to 10 5.3 EID 50 / ml (10 per chicken in 50 μl) 3 to 10 4 EID 50 ).

[0383] Example 4: Determination of vaccine efficacy

[0384] Fertilized SPF chicken eggs were incubated in an egg incubator at 99.7°F and 50% humidity for 18 days with hourly turning. On the 18th day of incubation, the eggs were candidized and the fertilized eggs were transferred to an incubator and incubated at 99°F and 70% humidity until hatched. Chickens without clinical signs or deformations were randomly assigned to treatment groups and moved to separate isolators. At least two chickens were used as a strict negative control (SNC) group, five chickens were included in the challenge control (CC) group, and at least ten chickens in each group were vaccinated with recombinant IBV with a heterologous spike protein or spike protein extracellular domain and subsequently challenged. Animals were housed under housing conditions that met local and national animal welfare recommendations. The lighting regimen was adjusted to 16 hours of light per day. Feed and water were provided ad libitum. After transfer to the isolator, chickens (1 day old) were inoculated with 10 3 EID 50 / , while the SNC and CC groups remained untreated. 21 days after vaccination, the chickens in the CC and vaccinated groups were treated with 10 3 to 10 4 EID 50 / were challenged with the respective spike protein-homologous challenge strain (793B, QX, Q1, Ark, variant 2 or Brazil). On the 7th day after the challenge, all chickens were euthanized, pharyngeal swabs were taken, kidneys were removed, and stored in RNAlater stabilization solution (ThermoFisher) at 4°C for IBV-specific RT-qPCR analysis. In addition, the trachea was removed and moved to a 50 ml test tube filled with warm cell culture medium. The trachea was then cleared from the connective tissue and rinsed with cell culture medium. The trachea was cut into tracheal rings using a McIlwain tissue chopper set to 0.6-0.8 mm slice thickness. Three rings in the upper part of each trachea, four rings in the middle part, and three rings in the lower part were analyzed by light microscopy and scored for ciliary arrest (see Table 5). If more than 50% of the internal rings showed obvious ciliary movement (score 2 and lower), it was recorded as normal. If less than 50% of the cilia were motile (scores 3 and 4), the ring was scored as positive for ciliary arrest.

[0385] For IBV-specific RT-qPCR analysis, kidney tissue sections were warmed to room temperature and then transferred to separate 2 ml Precellys tubes filled with culture medium and PBS, respectively. The kidneys were homogenized at 6800 rpm for 1 x 20 seconds using a tissue homogenizer (Bertin Instruments). Throat swabs were eluted with 2 ml of 1 x PBS. MagMAX TM Core Nucleic Acid Purification Kit (ThermoFisher) and KingFisher TM The Duo Prime purification system (ThermoFisher) was used to separate nucleic acids from 200 μl of eluate and tissue homogenate. TM RT-qPCR was performed as described above for in ovo kinetics, except that analysis was performed in duplicate using a Real-Time PCR system (ThermoFisher).

[0386] Table 5: Ciliary arrest scores in tracheal rings

[0387] Cilia activity [%] Ciliary arrest score 100 0 <100-75 1 <75-50 2 <50-25 3 <25-0 4

[0388] Example 5: Efficacy of recombinant IBV H52 encoding heterologous spike protein or spike protein ectodomain

[0389] The aim of this study was to demonstrate that vaccination with recombinant IBV H52 (Mass genotype) encoding a heterologous spike protein or spike protein extracellular domain confers protection against challenge with a spike protein homologous challenge strain.

[0390] The study analyzed whether recombinant IBV H52 (Mass genotype) encoding the extracellular domain of the IBV CR88 (4 / 91 genotype) spike protein could confer protection against challenge with the virulent 793B strain (4 / 91 genotype), which is considered to be a homologous challenger to the encoded IBV CR88 spike protein extracellular domain and a heterologous challenger to the IBV H52 backbone. All chickens were observed daily for clinical signs. No clinical signs were recorded after vaccination or challenge. Back titration of the inoculation with H52 rIBV CR88 S Etco at 1 day of age determined that the titers of the 793B challenge virus applied on day 21 after vaccination were 10 4.13 EID 50 / ml(target 10 4.3 EID 50 / ml), and 10 4.69 EID 50 / ml(target 10 4.3 EID 50 Ciliary motility arrest was scored as described above. Figure 2 are shown and summarized in Table 6.

[0391] Table 6: Overview of the ciliary arrest protection scores at 28 days post-vaccination and 7 days post-challenge. The average ciliary arrest score for each group was calculated by adding the total scores of each chicken in each group and then dividing the group total score by the number of animals (maximum score is 40 and minimum score is 0). If no fewer than 9 out of 10 rings showed normal ciliary activity, the animal was considered unaffected.

[0392]

[0393] All animals in the strict negative control group showed normal ciliary motility, while 80% of animals in the challenge control group were positive for ciliary arrest. In contrast, 82% of animals vaccinated with H52 rIBV CR88 S Etco were protected. In addition, viral loads in kidney and nasal swabs of animals vaccinated with H52 rIBV CR88 S Etco were reduced compared with animals in the challenge control group ( Figure 3 and Figure 4 ).

[0394] In addition, we analyzed whether recombinant IBV H52 encoding the IBV QX spike protein could confer protection against challenge with the virulent D388QX strain, as a homologous challenge agent encoding the IBV QX spike protein, and as a heterologous challenge agent to the IBV H52 backbone. All birds were observed daily for clinical signs. No clinical signs were recorded after vaccination or challenge. The titer, determined by reverse titration with CH52 rIBV QX S at 1 day of age, was 10 4 EID 50 / ml, while QX vaccination exceeds 10 5 EID 50 / ml(target 10 4.3 EID 50 / ml). The titer of the virus was 10 at 21 days after inoculation when challenged with D388 QX. 4.83 EID 50 / ml(target 10 4.3 EID 50 / ml). Ciliary arrest was scored as described above, and the results were as follows. Figure 5 are shown and summarized in Table 7.

[0395] Table 7: Overview of the ciliary arrest protection scores at 28 days post-vaccination and 7 days post-challenge. The average ciliary arrest score for each group was calculated by adding the total scores of each chicken in each group and then dividing the group total score by the number of animals (maximum score is 40 and minimum score is 0). If no less than 9 out of 10 rings showed normal ciliary activity, the animal was considered unaffected.

[0396] Group vaccine attack Mean ciliary arrest score Not affected [%] 1 - - 0 100 2 - D388 QX 38.4 0 3 QX vaccine D388 QX 3.5 100 3 H52 rIBV QX S D388 QX 5.9 100

[0397] All animals in the strict negative control group showed normal ciliary motility, while all animals in the challenge control group tested positive for ciliary arrest. In contrast, 100% of animals vaccinated with the H52 rIBV QX S or QX vaccine were protected.

[0398] Similar results were obtained with other H52 rIBVs with heterologous spike proteins or spike protein extracellular domains.

[0399] This result highlights the suitability of IBV H52 as an efficient backbone for generating recombinant IBV with heterologous spike proteins, particularly when compared to prior art data for the IBV Beaudette backbone showing excellent results. Sequence Listing <110> Boehringer Ingelheim Animal Health GmbH <120> H52 IBV vaccine with heterologous spike protein <130> 01-3344 <160> 85 <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 Rec 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[[ID=)28]] <210> 2 <211> 409 <212> PRT <213> Avian infectious bronchitis virus <400> 2 Met Ala Ser Gly Lys Thr Thr Gly Lys Thr Asp Ala Pro Ala Pro Val 1 5 10 15 Ile Lys Leu Gly Gly Pro Lys Pro Pro Lys Val Gly Ser Ser Gly Asn 20 25 30 Ala Ser Trp Phe Gln Ala Leu Lys Ala Lys Lys Leu Asn Ser Pro Pro 35 40 45 Pro Lys Phe Glu Gly Ser Gly Val Pro Asp Asn Glu Asn Leu Lys Leu 50 55 60 Ser Gln Gln His Gly Tyr Trp Arg Arg Gln Ala Arg Tyr Lys Pro Gly 65 70 75 80 Lys Gly Gly Arg Lys Ser Val Pro Asp Ala Trp Tyr Phe Tyr Tyr Thr 85 90 95 Gly Thr Gly Pro Ala Ala Asp Leu Asn Trp Gly Asp Ser Gln Asp Gly 100 105 110 Ile Val Trp Val Ser Ala Lys Gly Ala Asp Thr Lys Ser Arg Ser Asn 115 120 125 Gln Gly Thr Arg Asp Pro Asp Lys Phe Asp Gln Tyr Pro Leu Arg Phe 130 135 140 Ser Asp Gly Gly Pro Asp Gly Asn Phe Arg Trp Asp Phe Ile Pro Ile 145 150 155 160 Asn Arg Gly Arg Ser Gly Arg Ser Thr Ala Ala Ser Ser Ala Ala Ser 165 170 175 Ser Arg Ala Pro Ser Arg Asp Gly Ser Arg Gly Arg Arg Ser Gly Ala 180 185 190 Glu Asp Asp Leu Ile Ala Arg Ala Ala Lys Ile Ile Gln Asp Gln Gln 195 200 205 Lys Lys Gly Ser Arg Ile Thr Lys Ala Lys Ala Asp Glu Met Ala His 210 215 220 Arg Arg Tyr Cys Lys Arg Thr Ile Pro Pro Gly Tyr Lys Val Asp Gln 225 230 235 240 Val Phe Gly Pro Arg Thr Lys Gly Lys Glu Gly Asn Phe Gly Asp Asp 245 250 255 Lys Met Asn Glu Glu Gly Ile Lys Asp Gly Arg Val Thr Ala Met Leu 260 265 270 Asn Leu Val Pro Ser Ser His Ala Cys Leu Phe Gly Ser Arg Val Thr 275 280 285 Pro Lys Leu Gln Pro Asp Gly Leu His Leu Arg Phe Glu Phe Thr Thr 290 295 300 Val Val Ser Arg Asp Asp Pro Gln Phe Asp Asn Tyr Val Lys Ile Cys 305 310 315 320 Asp Gln Cys Val Asp Gly Val Gly Thr Arg Pro Lys Asp Asp Glu Pro 325 330 335 Arg Pro Lys Ser Arg Pro Asn Ser Arg Pro Ala Thr Arg Thr Ser Ser 340 345 350 Pro Ala Pro Arg Gln Gln Arg Gln Lys Lys Glu Lys Lys Ser Lys Lys 355 360 365 Gln Asp Asp Glu Val Asp Lys Ala Leu Thr Ser Asp Glu Glu Arg Asn 370 375 380 Asn Ala Gln Leu Glu Phe Asp Asp Glu Pro Lys Val Ile Asn Trp Gly 385 390 395 400 Asp Ser Ala Leu Gly Glu Asn Glu Leu 405 <210> 3 <211> 110 <212> PRT <213> Avian infectious bronchitis virus <400> 3 Val Met Met Asn Leu Leu Asn Lys Ser Leu Glu Glu Asn Gly Ser Phe 1 5 10 15 Leu Thr Ala Leu Tyr Ile Phe Val Gly Phe Leu Ala Phe Tyr Leu Leu 20 25 30 Gly Arg Ala Leu Gln Ala Phe Val Gln Ala Ala Asp Ala Cys Cys Leu 35 40 45 Phe Trp Tyr Thr Trp Leu Val Ile Pro Gly Val Lys Gly Thr Ala Phe 50 55 60 Val Tyr Lys Tyr Thr Tyr Gly Arg Lys Leu Asn Asn Ser Glu Leu Glu 65 70 75 80 Ala Val Val Val Asn Glu Phe Pro Lys Asn Gly Trp Asn Asn Lys Asn 85 90 95 Pro Ala Asn Phe Gln Asp Val Gln Arg Asn Lys Leu Tyr Ser 100 105 110 <210> 4 <211> 225 <212> PRT <213> Avian infectious bronchitis virus <400> 4 Met Ser Asn Glu Thr Asn Cys Thr Leu Asp Phe Glu Gln Ser Val Glu 1 5 10 15 Leu Phe Lys Glu Tyr Asn Leu Phe Ile Thr Ala Phe Leu Leu Phe Leu 20 25 30 Thr Ile Ile Leu Gln Tyr Gly Tyr Ala Thr Arg Ser Lys Phe Ile Tyr 35 40 45 Ile Leu Lys Met Ile Val Leu Trp Cys Phe Trp Pro Leu Asn Ile Ala 50 55 60 Val Gly Val Ile Ser Cys Ile Tyr Pro Pro Asn Thr Gly Gly Leu Val 65 70 75 80 Ala Ala Ile Ile Leu Thr Val Phe Ala Cys Leu Ser Phe Val Gly Tyr 85 90 95 Trp Ile Gln Ser Ile Arg Leu Phe Lys Arg Cys Arg Ser Trp Trp Ser 100 105 110 Phe Asn Pro Glu Ser Asn Ala Val Gly Ser Ile Leu Leu Thr Asn Gly 115 120 125 Gln Gln Cys Asn Phe Ala Ile Glu Ser Val Pro Met Val Leu Ser Pro 130 135 140 Ile Ile Lys Asn Gly Val Leu Tyr Cys Glu Gly Gln Trp Leu Ala Lys 145 150 155 160 Cys Glu Pro Asp His Leu Pro Lys Asp Ile Phe Val Cys Thr Pro Asp 165 170 175 Arg Arg Asn Ile Tyr Arg Met Val Gln Lys Tyr Thr Gly Asp Gln Ser 180 185 190 Gly Asn Lys Lys Arg Phe Ala Thr Phe Val Tyr Ala Lys Gln Ser Val 195 200 205 Asp Thr Gly Glu Leu Glu Ser Val Ala Thr Gly Gly Ser Ser Leu Tyr 210 215 220 Thr 225 <210> 5 <211> 1155 <212> PRT <213> Avian infectious bronchitis virus <400> 5 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> 6 <211> 1077 <212> PRT <213> Avian infectious bronchitis virus <400> 6 Leu Leu Tyr Asp Asn Asn Thr Tyr Val Tyr Tyr Tyr Gln Ser Ala Phe 1 5 10 15 Arg Pro Gly Pro Gly Trp His Leu Tyr Gly Gly Ala Tyr Ala Val Asp 20 25 30 Arg Val Phe Asn Glu Thr Asn Asn Ala Gly Ser Ala Ser Asp Cys Thr 35 40 45 Ala Gly Thr Phe Tyr Glu Ser His Asn Ile Ser Ala Ser Ser Val Ala 50 55 60 Met Thr Val Pro His Asn Gly Met Ser Trp Ser Ala Ser Gln Phe Cys 65 70 75 80 Thr Ala His Cys Asn Phe Ser Asp Phe Thr Val Phe Val Thr His Cys 85 90 95 Phe Lys Asn Gln Leu Gly Ser Cys Pro Leu Thr Gly Met Ile Pro Gln 100 105 110 Asn His Ile Arg Ile Ser Ala Met Arg Asp Gly Val Leu Phe Tyr Asn 115 120 125 Leu Thr Val Ser Val Ser Lys Tyr Pro Arg Phe Lys Ser Leu Gln Cys 130 135 140 Val Ser Asn Ser Thr Ser Val Tyr Val Asn Gly Asp Leu Val Phe Thr 145 150 155 160 Ser Asn Glu Thr Ser Tyr Val Thr Gly Ala Gly Val Tyr Phe Lys Ser 165 170 175 Gly Gly Pro Val Thr Tyr Lys Val Met Lys Glu Val Lys Ala Leu Ala 180 185 190 Tyr Phe Ile Asn Gly Thr Ala Gln Glu Val Ile Leu Cys Asp Asn Ser 195 200 205 Pro Arg Gly Leu Leu Ala Cys Gln Tyr Asn Thr Gly Asn Phe Ser Asp 210 215 220 Gly Phe Tyr Pro Phe Thr Asn His Ser Leu Val Lys Asp Arg Phe Ile 225 230 235 240 Val Tyr Arg Glu Ser Ser Thr Asn Thr Thr Leu Lys Leu Thr Asn Phe 245 250 255 Ser Phe Thr Asn Val Ser Asn Ala Ser Pro Asn Ser Gly Gly Val Asp 260 265 270 Thr Phe Gln Leu Tyr Gln Thr Ser Thr Ala Gln Asp Gly Tyr Tyr Asn 275 280 285 Phe Asn Leu Ser Phe Leu Ser Ser Phe Val Tyr Lys Pro Ser Asp Phe 290 295 300 Met Tyr Gly Ser Tyr His Pro His Cys Lys Phe Arg Pro Glu Asn Ile 305 310 315 320 Asn Asn Gly Leu Trp Phe Asn Ser Leu Ser Val Ser Leu Thr Tyr Gly 325 330 335 Pro Ile Gln Gly Gly Cys Lys Gln Ser Val Phe Ser Asn Arg Ala Thr 340 345 350 Cys Cys Tyr Ala Tyr Ser Tyr Gln Gly Pro Ser Arg Cys Lys Gly Val 355 360 365 Tyr Arg Gly Glu Leu Thr Gln Tyr Phe Glu Cys Gly Leu Leu Val Tyr 370 375 380 Val Thr Lys Ser Asp Gly Ser Arg Ile Gln Thr Arg Ser Glu Pro Leu 385 390 395 400 Val Leu Thr Gln Tyr Asn Tyr Asn Asn Ile Thr Leu Asn Lys Cys Val 405 410 415 Glu Tyr Asn Ile Tyr Gly Arg Val Gly Gln Gly Phe Ile Thr Asn Val 420 425 430 Thr Glu Ala Thr Ala Asn Tyr Ser Tyr Leu Ala Asp Gly Gly Leu Ala 435 440 445 Ile Leu Asp Thr Ser Gly Ala Ile Asp Ile Phe Val Val Gln Gly Ala 450 455 460 Tyr Gly Leu Asn Tyr Tyr Lys Val Asn Pro Cys Glu Asp Val Asn Gln 465 470 475 480 Gln Phe Val Val Ser Gly Gly Asn Leu Val Gly Ile Leu Thr Ser His 485 490 495 Asn Glu Thr Gly Ser Glu Ser Ile Glu Asn Gln Phe Tyr Ile Lys Leu 500 505 510 Thr Asn Gly Thr Arg Arg Ser Arg Arg Ser Val Thr Gly Asn Val Thr 515 520 525 Asn Cys Pro Tyr Val Ser Tyr Gly Lys Phe Cys Ile Lys Pro Asp Gly 530 535 540 Ser Leu Ser Ile Ile Val Pro Gln Glu Leu Glu Gln Phe Val Ala Pro 545 550 555 560 Leu Phe Asn Val Thr Glu His Val Leu Ile Pro Asp Ser Phe Asn Leu 565 570 575 Thr Val Thr Asp Glu Tyr Ile Gln Thr Arg Met Asp Lys Val Gln Ile 580 585 590 Ile Cys Leu Gln Tyr Val Cys Gly Asn Ser Ile Glu Cys Arg Lys Leu 595 600 605 Phe Gln Gln Tyr Gly Pro Val Cys Asp Asn Ile Leu Ser Val Val Asn 610 615 620 Gly Val Gly Gln Arg Glu Asp Met Glu Leu Leu Ser Phe Tyr Ser Ser 625 630 635 640 Thr Lys Pro Ser Gly Tyr Asn Thr Pro Ile Phe Asn Asn Val Ser Thr 645 650 655 Gly Asp Phe Asn Ile Ser Leu Leu Leu Thr Pro Pro Asn Ser Pro Thr 660 665 670 Gly Arg Ser Phe Ile Glu Asp Leu Leu Phe Thr Ser Val Glu Ser Val 675 680 685 Gly Leu Pro Thr Asp Glu Glu Tyr Lys Lys Cys Thr Ala Gly Pro Leu 690 695 700 Gly Phe Val Lys Asp Leu Val Cys Ala Arg Glu Tyr Asn Gly Leu Leu 705 710 715 720 Val Leu Pro Pro Ile Ile Thr Ala Glu Met Gln Thr Met Tyr Thr Ser 725 730 735 Ser Leu Val Ala Ser Met Ala Leu Gly Gly Ile Thr Ala Ala Gly Ala 740 745 750 Ile Pro Phe Ala Thr Gln Leu Gln Ala Arg Ile Asn His Leu Gly Ile 755 760 765 Thr Asn Ser Leu Leu Leu Lys Asn Gln Glu Lys Ile Ala Ala Ser Phe 770 775 780 Asn Lys Ala Ile Gly His Met Gln Glu Gly Phe Lys Ser Thr Ser Leu 785 790 795 800 Ala Leu Gln Gln Ile Gln Asp Val Val Asn Lys Gln Ser Ser Ile Leu 805 810 815 Thr Glu Thr Met Gln Ser Leu Asn Lys Asn Phe Gly Ala Ile Ser Ser 820 825 830 Val Ile Gln Asp Ile Tyr Gln Gln Leu Asp Ala Ile Gln Ala Asp Ala 835 840 845 Gln Val Asp Arg Leu Ile Thr Gly Arg Leu Ser Ser Leu Ser Val Leu 850 855 860 Ala Ser Ala Lys Gln Ala Glu Tyr His Arg Val Ser Gln Gln Arg Glu 865 870 875 880 Leu Ala Thr Gln Lys Ile Asn Glu Cys Val Lys Ser Gln Ser Asn Arg 885 890 895 Tyr Ser Phe Cys Gly Asn Gly Arg His Val Leu Thr Ile Pro Gln Asn 900 905 910 Ala Pro Asn Gly Ile Val Phe Ile His Phe Thr Tyr Thr Pro Glu Ser 915 920 925 Phe Val Asn Val Thr Ala Ile Val Gly Phe Cys Val Asn Pro Ala Asn 930 935 940 Ala Ser His Tyr Ala Ile Val Pro Val Asn Gly Arg Gly Val Phe Ile 945 950 955 960 Glu Val Asn Gly Ser Tyr Tyr Ile Thr Ala Arg Asp Met Tyr Met Pro 965 970 975 Arg Asp Ile Thr Ala Gly Asp Ile Val Thr Leu Thr Ser Cys Gln Ala 980 985 990 Asn Tyr Val Asn Val Asn Lys Thr Val Ile Asn Thr Phe Val Glu Asp 995 1000 1005 Asp Asp Phe Asp Phe Tyr Asp Glu Leu Ser Lys Trp Trp Asn Asp 1010 1015 1020 Thr Lys His Glu Leu Pro Asp Phe Asp Glu Phe Asn Tyr Thr Val 1025 1030 1035 Pro Val Leu Asn Ile Ser Asn Glu Ile Asp Arg Ile Gln Gln Val 1040 1045 1050 Ile Gln Gly Leu Asn Asp Ser Leu Ile Asp Leu Glu Thr Leu Ser 1055 1060 1065 Ile Leu Lys Thr Tyr Ile Lys Trp Pro 1070 1075 <210> 7 <211> 1156 <212> PRT <213> Avian infectious bronchitis virus <400> 7 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 Gln 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> 8 <211> 1078 <212> PRT <213> Avian infectious bronchitis virus <400> 8 Asn Leu Phe Asp Ser Asp Asn Asn Tyr Val Tyr Tyr Tyr Gln Ser Ala 1 5 10 15 Phe Arg Pro Pro Asn Gly Trp His Leu Gln Gly Gly Ala Tyr Ala Val 20 25 30 Val Ser Ser Thr Asn Tyr Thr Asn Asn Ala Gly Ser Ala His Gly Cys 35 40 45 Thr Val Gly Val Ile Lys Asp Val Tyr Asn Gln Ser Val Ala Ser Ile 50 55 60 Ala Met Thr Ala Pro Leu Gln Gly Met Ala Trp Ser Lys Ser Gln Phe 65 70 75 80 Cys Ser Ala His Cys Asn Phe Ser Glu Ile Thr Val Phe Val Thr His 85 90 95 Cys Tyr Ser Ser Gly Ser Gly Ser Cys Pro Ile Thr Gly Met Ile Pro 100 105 110 Arg Asp His Ile Arg Ile Ser Ala Met Lys Asn Gly Ser Leu Phe Tyr 115 120 125 Asn Leu Thr Val Ser Val Ser Lys Tyr Pro Asn Phe Lys Ser Phe Gln 130 135 140 Cys Val Asn Asn Phe Thr Ser Val Tyr Leu Asn Gly Asp Leu Val Phe 145 150 155 160 Thr Ser Asn Lys Thr Thr Asp Val Thr Ser Ala Gly Val Tyr Phe Lys 165 170 175 Ala Gly Gly Pro Val Asn Tyr Ser Ile Met Lys Glu Phe Lys Val Leu 180 185 190 Ala Tyr Phe Val Asn Gly Thr Ala Gln Asp Val Val Leu Cys Asp Asn 195 200 205 Ser Pro Lys Gly Leu Leu Ala Cys Gln Tyr Asn Thr Gly Asn Phe Ser 210 215 220 Asp Gly Phe Tyr Pro Phe Thr Asn Ser Thr Leu Val Arg Glu Lys Phe 225 230 235 240 Ile Val Tyr Arg Glu Ser Ser Val Asn Thr Thr Leu Ala Leu Thr Asn 245 250 255 Phe Thr Phe Ser Asn Val Ser Asn Ala Gln Pro Asn Ser Gly Gly Val 260 265 270 Asn Thr Phe His Leu Tyr Gln Thr Gln Thr Ala Gln Ser Gly Tyr Tyr 275 280 285 Asn Phe Asn Leu Ser Phe Leu Ser Gln Phe Val Tyr Lys Ala Ser Asp 290 295 300 Phe Met Tyr Gly Ser Tyr His Pro Ser Cys Ser Phe Arg Pro Glu Thr 305 310 315 320 Ile Asn Ser Gly Leu Trp Phe Asn Ser Leu Ser Val Ser Leu Thr Tyr 325 330 335 Gly Pro Leu Gln Gly Gly Cys Lys Gln Ser Val Phe Ser Gly Lys Ala 340 345 350 Thr Cys Cys Tyr Ala Tyr Ser Tyr Arg Gly Pro Met Ala Cys Lys Gly 355 360 365 Val Tyr Ser Gly Glu Leu Ser Thr Asn Phe Glu Cys Gly Leu Leu Val 370 375 380 Tyr Val Thr Lys Ser Asp Gly Ser Arg Ile Gln Thr Arg Thr Glu Pro 385 390 395 400 Leu Val Leu Thr Gln Tyr Asn Tyr Asn Asn Ile Thr Leu Asp Lys Cys 405 410 415 Val Ala Tyr Asn Ile Tyr Gly Arg Val Gly Gln Gly Phe Ile Thr Asn 420 425 430 Val Thr Asp Ser Ala Ala Asn Phe Ser Tyr Leu Ala Asp Gly Gly Leu 435 440 445 Ala Ile Leu Asp Thr Ser Gly Ala Ile Asp Val Phe Val Val Gln Gly 450 455 460 Ile Tyr Gly Leu Asn Tyr Tyr Lys Val Asn Pro Cys Glu Asp Val Asn 465 470 475 480 Gln Gln Phe Val Val Ser Gly Gly Asn Ile Val Gly Ile Leu Thr Ser 485 490 495 Arg Asn Glu Thr Gly Ser Glu Gln Val Glu Asn Gln Phe Tyr Val Lys 500 505 510 Leu Thr Asn Ser Ser His Arg Arg Lys Arg Ser Ile Gly Gln Asn Val 515 520 525 Thr Ser Cys Pro Tyr Val Ser Tyr Gly Arg Phe Cys Ile Glu Pro Asp 530 535 540 Gly Ser Leu Lys Met Ile Val Pro Glu Glu Leu Lys Gln Phe Val Ala 545 550 555 560 Pro Leu Leu Asn Ile Thr Glu Ser Val Leu Ile Pro Asn Ser Phe Asn 565 570 575 Leu Thr Val Thr Asp Glu Tyr Ile Gln Thr Arg Met Asp Lys Val Gln 580 585 590 Ile Asn Cys Leu Gln Tyr Val Cys Gly Asn Ser Leu Glu Cys Arg Lys 595 600 605 Leu Phe Gln Gln Tyr Gly Pro Val Cys Asp Asn Ile Leu Ser Val Val 610 615 620 Asn Ser Val Ser Gln Lys Glu Asp Met Glu Leu Leu Ser Phe Tyr Ser 625 630 635 640 Ser Thr Lys Pro Lys Gly Tyr Asp Thr Pro Val Leu Ser Asn Val Ser 645 650 655 Thr Gly Glu Phe Asn Ile Ser Leu Leu Leu Lys Pro Pro Ser Ser Pro 660 665 670 Ser Gly Arg Ser Phe Ile Glu Glu Leu Leu Phe Thr Ser Val Glu Thr 675 680 685 Val Gly Leu Pro Thr Asp Ala Glu Tyr Lys Lys Cys Thr Ala Gly Pro 690 695 700 Leu Gly Thr Leu Lys Asp Leu Ile Cys Ala Arg Glu Tyr Asn Gly Leu 705 710 715 720 Leu Val Leu Pro Pro Ile Ile Thr Ala Asp Met Gln Thr Met Tyr Thr 725 730 735 Ala Ser Leu Val Gly Ala Met Ala Phe Gly Gly Ile Thr Ser Ala Ala 740 745 750 Ala Ile Pro Phe Ala Thr Gln Ile Gln Ala Arg Ile Asn His Leu Gly 755 760 765 Ile Thr Gln Ser Leu Leu Met Lys Asn Gln Glu Lys Ile Ala Ala Ser 770 775 780 Phe Asn Lys Ala Ile Gly His Met Gln Glu Gly Phe Arg Ser Thr Ser 785 790 795 800 Leu Ala Leu Gln Gln Ile Gln Asp Val Val Asn Lys Gln Ser Ala Ile 805 810 815 Leu Thr Glu Thr Met Asn Ser Leu Asn Lys Asn Phe Gly Ala Ile Thr 820 825 830 Ser Val Ile Gln Asp Ile Tyr Ala Gln Leu Asp Ala Ile Gln Ala Asp 835 840 845 Ala Gln Val Asp Arg Leu Ile Thr Gly Arg Leu Ser Ser Leu Ser Val 850 855 860 Leu Ala Ser Ala Lys Gln Ser Glu Tyr Ile Arg Val Ser Gln Gln Arg 865 870 875 880 Glu Leu Ala Thr Gln Lys Ile Asn Glu Cys Val Lys Ser Gln Ser Asn 885 890 895 Arg Tyr Gly Phe Cys Gly Ser Gly Arg His Val Leu Ser Ile Pro Gln 900 905 910 Asn Ala Pro Asn Gly Ile Val Phe Ile His Phe Thr Tyr Thr Pro Glu 915 920 925 Ser Phe Val Asn Val Thr Ala Ile Val Gly Phe Cys Val Asn Pro Ala 930 935 940 Asn Ala Ser Gln Tyr Ala Ile Val Pro Ala Asn Gly Arg Gly Ile Phe 945 950 955 960 Ile Gln Val Asn Gly Thr Tyr Tyr Ile Thr Ala Arg Asp Met Tyr Met 965 970 975 Pro Arg Asp Ile Thr Ala Gly Asp Ile Val Thr Leu Thr Ser Cys Gln 980 985 990 Ala Asn Tyr Val Asn Val Asn Lys Thr Val Ile Thr Thr Phe Val Glu 995 1000 1005 Asp Asp Asp Phe Asp Phe Asp Asp Glu Leu Ser Lys Trp Trp Asn 1010 1015 1020 Asp Thr Lys His Gln Leu Pro Asp Phe Asp Asp Phe Asn Tyr Thr 1025 1030 1035 Val Pro Ile Leu Asn Ile Ser Gly Glu Ile Asp Tyr Ile Gln Gly 1040 1045 1050 Val Ile Gln Gly Leu Asn Asp Ser Leu Ile Asp Leu Glu Glu Leu 1055 1060 1065 Ser Ile Ile Lys Thr Tyr Ile Lys Trp Pro 1070 1075 <210> 9 <211> 1166 <212> PRT <213> Avian infectious bronchitis virus <400> 9 Met Leu Gly 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 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 Glu Thr Asn Asn Ala Gly Thr Ala Ser Gln 50 55 60 Cys Ile Ala Gly Ala Ile Ser Trp Ser Lys Asn Phe Ser Ala Ser Ala 65 70 75 80 Val Ala Met Thr Ala Pro Glu Leu Gly Met Thr Trp Ser Thr Gly 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 Asn Gly Leu Cys Pro Leu Thr Gly Leu 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 Thr Met Ser 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 Thr Leu Val 260 265 270 Leu Thr Asn Phe Thr Phe Ser Asn Val Ser Asn Ala Pro Pro Asn Thr 275 280 285 Gly Gly Val His Ser Ile Val Leu His Gln Thr Gln Thr Ala Gln Ser 290 295 300 Gly Tyr Tyr Asn Phe Asn Phe Ser Phe Leu Ser Ser 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 Leu 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 Met Ala Thr Cys Cys Tyr Ala Tyr Ser Tyr Ser Gly Pro Thr Leu 370 375 380 Cys Lys Gly Val Tyr Ser Gly Gln 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 Thr 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 Pro 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 Ser 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 Arg 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 Ile 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 Gln 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 Arg Ile Gln Glu Val Ile Gln 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 Thr Glu 1145 1150 1155 Gln Tyr Arg Pro Lys Lys Ser Val 1160 1165 <210> 10 <211> 1079 <212> PRT <213> Avian infectious bronchitis virus <400> 10 Ala Leu Phe Asp Asn Asn Glu Thr Val Tyr Tyr Tyr Gln Ser Ala Phe 1 5 10 15 Arg Pro Ala Asp Gly Trp His Leu His Gly Gly Ala Tyr Ala Val Val 20 25 30 Asn Val Ser Leu Glu Thr Asn Asn Ala Gly Thr Ala Ser Gln Cys Ile 35 40 45 Ala Gly Ala Ile Ser Trp Ser Lys Asn Phe Ser Ala Ser Ala Val Ala 50 55 60 Met Thr Ala Pro Glu Leu Gly Met Thr Trp Ser Thr Gly Gln Phe Cys 65 70 75 80 Thr Ala His Cys Asn Phe Ser Asp Phe Thr Val Phe Val Thr His Cys 85 90 95 Phe Lys His Gly Asn Gly Leu Cys Pro Leu Thr Gly Leu Ile Pro Ser 100 105 110 Gly Phe Ile Arg Val Ser Ala Met Arg Lys Gly Ser Asn Ser Leu Phe 115 120 125 Tyr Asn Leu Thr Val Ser Val Thr Lys Tyr Pro Arg Phe Lys Ser Leu 130 135 140 Gln Cys Val Asn Asn Tyr Thr Ser Val Tyr Leu Asn Gly Asp Leu Val 145 150 155 160 Phe Thr Ser Asn Glu Thr Lys Pro Val Ser Ala Ala Gly Val Ser Phe 165 170 175 Lys Ala Gly Gly Pro Ile Thr Tyr Lys Thr Met Ser Glu Val Lys Val 180 185 190 Leu Ala Tyr Phe Val Asn Gly Thr Ala Gln Thr Val Ile Pro Cys Asp 195 200 205 Gly Ser Pro Arg Gly Leu Leu Ala Cys Gln Tyr Asn Thr Gly Asn Phe 210 215 220 Ser Asp Gly Phe Tyr Pro Tyr Thr Asn Ser Ser Leu Val Lys Glu Arg 225 230 235 240 Phe Ile Val Tyr Arg Glu Ser Ser Val Asn Thr Thr Leu Val Leu Thr 245 250 255 Asn Phe Thr Phe Ser Asn Val Ser Asn Ala Pro Pro Asn Thr Gly Gly 260 265 270 Val His Ser Ile Val Leu His Gln Thr Gln Thr Ala Gln Ser Gly Tyr 275 280 285 Tyr Asn Phe Asn Phe Ser Phe Leu Ser Ser Phe Arg Tyr Val Glu Ser 290 295 300 Asp Phe Met Tyr Gly Ser Tyr His Pro Lys Cys Ser Phe Arg Leu Glu 305 310 315 320 Thr Ile Asn Asn Gly Leu Trp Phe Asn Ser Leu Ser Val Ser Leu Gly 325 330 335 Tyr Gly Pro Leu Gln Gly Gly Cys Lys Gln Ser Val Phe Asn Asn Met 340 345 350 Ala Thr Cys Cys Tyr Ala Tyr Ser Tyr Ser Gly Pro Thr Leu Cys Lys 355 360 365 Gly Val Tyr Ser Gly Gln Leu Gln Lys Thr Phe Glu Cys Gly Leu Leu 370 375 380 Val Phe Val Thr Lys Ser Asp Gly Ser Arg Ile Gln Thr Arg Asn Glu 385 390 395 400 Pro Leu Val Leu Thr Gln His Asn Tyr Asn Asn Ile Thr Leu Asn Lys 405 410 415 Cys Val Glu Tyr Asn Ile Tyr Gly Arg Val Gly Gln Gly Leu Ile Thr 420 425 430 Asn Ile Thr Asp Ser Ala Ala Asn His Gly Tyr Leu Ala Asp Gly Gly 435 440 445 Leu Ala Val Leu Asp Thr Ser Gly Ala Ile Asp Val Phe Val Val Gln 450 455 460 Gly Val Tyr Gly Leu Thr Tyr Tyr Lys Val Asn Pro Cys Glu Asp Val 465 470 475 480 Asn Gln Gln Phe Val Val Ser Gly Gly Gln Leu Val Gly Ile Leu Thr 485 490 495 Ser Arg Asn Glu Thr Gly Ser Gln Pro Ile Glu Asn Arg Phe Tyr Val 500 505 510 Lys Phe Pro Asn Ser Arg Arg Arg Thr Gly Arg Ser Thr Ile Ala Asn 515 520 525 Val Thr Asn Cys Pro Tyr Val Ser Tyr Gly Lys Phe Cys Ile Lys Pro 530 535 540 Asp Gly Ser Val Ser Glu Ile Val Pro Gln Glu Ile Glu His Phe Val 545 550 555 560 Ala Pro Leu Leu Asn Val Thr Glu His Val Leu Ile Pro Asn Ser Phe 565 570 575 Asn Leu Thr Val Thr Asp Glu Tyr Ile Gln Thr Arg Met Asp Lys Ile 580 585 590 Gln Ile Asn Cys Arg Gln Tyr Val Cys Gly Asn Ser Ile Glu Cys Arg 595 600 605 Lys Leu Phe Gln Gln Tyr Gly Pro Val Cys Asp Asn Ile Leu Ser Val 610 615 620 Val Asn Thr Val Gly Gln Arg Glu Asp Met Glu Leu Leu Ser Phe Tyr 625 630 635 640 Ser Ser Thr Lys Pro Lys Asp Tyr Asn Ile Pro Ile Phe Ser Asn Val 645 650 655 Ser Thr Gly Asp Phe Asn Ile Ser Leu Leu Leu Thr Pro Pro Asn Ser 660 665 670 Pro Thr Gly Arg Ser Phe Ile Glu Asp Ile Leu Phe Thr Ser Val Glu 675 680 685 Ser Val Gly Leu Pro Thr Asp Glu Glu Tyr Lys Lys Cys Thr Ala Gly 690 695 700 Pro Leu Gly Phe Val Lys Asp Leu Val Cys Ala Arg Glu Tyr Asn Gly 705 710 715 720 Leu Leu Val Leu Pro Pro Ile Ile Thr Ala Asp Met Gln Thr Met Tyr 725 730 735 Thr Ser Thr Leu Val Ala Ser Met Ala Leu Gly Gly Ile Thr Ala Ala 740 745 750 Gly Ala Ile Pro Phe Ala Thr Gln Leu Gln Ala Arg Ile Asn His Leu 755 760 765 Gly Ile Thr Gln Ser Leu Leu Leu Lys Asn Gln Glu Lys Ile Ala Ala 770 775 780 Ser Phe Asn Lys Ala Ile Gly His Met Gln Glu Gly Phe Arg Ser Thr 785 790 795 800 Ser Leu Ala Leu Gln Gln Ile Gln Asp Val Val Asn Lys Gln Ser Ser 805 810 815 Ile Leu Thr Glu Thr Met Ala Ser Leu Asn Lys Asn Phe Gly Ala Ile 820 825 830 Ser Ser Val Leu Gln Asp Ile Tyr Gln Gln Leu Asp Ala Ile Gln Ala 835 840 845 Asp Ala Gln Val Asp Arg Ile Ile Thr Gly Arg Leu Ser Ser Leu Ser 850 855 860 Val Leu Ala Ser Ala Lys Gln Ser Glu Tyr Tyr Arg Val Ser Gln Gln 865 870 875 880 Arg Glu Leu Ala Thr Gln Lys Ile Asn Glu Cys Val Lys Ser Gln Ser 885 890 895 Thr Arg Tyr Ser Phe Cys Gly Asn Gly Arg His Val Leu Thr Ile Pro 900 905 910 Gln Asn Ala Pro Asn Gly Ile Val Phe Ile His Phe Thr Tyr Thr Pro 915 920 925 Glu Ser Phe Val Asn Val Thr Ala Ile Val Gly Phe Cys Val Asn Pro 930 935 940 Pro Asn Ala Ser Gln Tyr Ala Leu Val Pro Ala Asn Gly Arg Gly Ile 945 950 955 960 Phe Ile Gln Val Asn Gly Ser Tyr Tyr Ile Thr Ala Arg Asp Met Tyr 965 970 975 Met Pro Arg Asp Ile Thr Ala Gly Asp Ile Val Thr Leu Thr Ser Cys 980 985 990 Gln Ala Asn Tyr Val Ser Val Asn Arg Thr Val Ile Thr Thr Phe Val 995 1000 1005 Asp Asn Asp Asp Phe Asp Phe Asp Asp Glu Leu Ser Lys Trp Trp 1010 1015 1020 Asn Asp Thr Lys His Glu Leu Pro Asp Phe Asp Glu Phe Asn Tyr 1025 1030 1035 Thr Ile Pro Val Leu Asn Ile Ser Asn Glu Ile Asp Arg Ile Gln 1040 1045 1050 Glu Val Ile Gln Gly Leu Asn Asp Ser Leu Ile Asp Leu Glu Ala 1055 1060 1065 Leu Ser Ile Leu Lys Thr Tyr Ile Lys Trp Pro 1070 1075 <210> 11 <211> 1159 <212> PRT <213> infectious diseases <400> 11 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 Gln 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> 12 <211> 1081 <212> PRT <213> Avian infectious bronchitis virus <400> 12 Asn Leu Tyr Asp Asn Glu Ser Phe Val Tyr Tyr Tyr Gln Ser Ala Phe 1 5 10 15 Arg Pro Gly His Gly Trp His Leu His Gly Gly Ala Tyr Ala Val Val 20 25 30 Asn Val Ser Ser Glu Asn Asn Asn Ala Gly Thr Ala Pro Ser Cys Thr 35 40 45 Ala Gly Ala Ile Gly Tyr Ser Lys Asn Phe Ser Ala Ala Ser Val Ala 50 55 60 Met Thr Ala Pro Leu Ser Gly Met Ser Trp Ser Ala Ser Ser Phe Cys 65 70 75 80 Thr Ala His Cys Asn Phe Thr Ser Tyr Ile Val Phe Val Thr His Cys 85 90 95 Phe Lys Ser Gly Ser Asn Ser Cys Pro Leu Thr Gly Leu Ile Pro Ser 100 105 110 Gly Tyr Ile Arg Ile Ala Ala Met Lys His Gly Ser Arg Thr Pro Gly 115 120 125 His Leu Phe Tyr Asn Leu Thr Val Ser Val Thr Lys Tyr Pro Lys Phe 130 135 140 Arg Ser Leu Gln Cys Val Asn Asn His Thr Ser Val Tyr Leu Asn Gly 145 150 155 160 Asp Leu Val Phe Thr Ser Gly Tyr Thr Glu Asp Val Val Ala Ala Gly 165 170 175 Val His Phe Lys Ser Gly Gly Pro Ile Thr Tyr Lys Val Met Arg Glu 180 185 190 Val Lys Ala Leu Ala Tyr Phe Val Asn Gly Thr Ala His Asp Val Ile 195 200 205 Leu Cys Asp Asp Thr Pro Arg Gly Leu Leu Ala Cys Gln Tyr Asn Thr 210 215 220 Gly Asn Phe Ser Asp Gly Phe Tyr Pro Phe Thr Asn Thr Ser Ile Val 225 230 235 240 Lys Asp Lys Phe Ile Val Tyr Arg Glu Ser Ser Val Asn Thr Thr Leu 245 250 255 Thr Leu Thr Asn Phe Thr Phe Ser Asn Glu Ser Gly Ala Pro Pro Asn 260 265 270 Thr Gly Gly Val Asp Ser Phe Ile Leu Tyr Gln Thr Gln Thr Ala Gln 275 280 285 Ser Gly Tyr Tyr Asn Phe Asn Phe Ser Phe Leu Ser Ser Phe Val Tyr 290 295 300 Arg Glu Ser Asn Tyr Met Tyr Gly Ser Tyr His Pro Arg Cys Ser Phe 305 310 315 320 Arg Pro Glu Thr Leu Asn Gly Leu Trp Phe Asn Ser Leu Ser Val Ser 325 330 335 Leu Thr Tyr Gly Pro Ile Gln Gly Gly Cys Lys Gln Ser Val Phe Asn 340 345 350 Gly Lys Ala Thr Cys Cys Tyr Ala Tyr Ser Tyr Gly Gly Pro Arg Ala 355 360 365 Cys Lys Gly Val Tyr Arg Gly Glu Leu Thr Gln His Phe Glu Cys Gly 370 375 380 Leu Leu Val Tyr Val Thr Lys Ser Asp Gly Ser Arg Ile Gln Thr Ala 385 390 395 400 Thr Gln Pro Pro Val Leu Thr Gln Asn Phe Tyr Asn Asn Ile Thr Leu 405 410 415 Gly Lys Cys Val Asp Tyr Asn Val Tyr Gly Arg Thr Gly Gln Gly Phe 420 425 430 Ile Thr Asn Val Thr Asp Leu Ala Thr Ser His Asn Tyr Leu Ala Asp 435 440 445 Gly Gly Leu Ala Ile Leu Asp Thr Ser Gly Ala Ile Asp Ile Phe Val 450 455 460 Val Gln Gly Glu Tyr Gly Pro Asn Tyr Tyr Lys Val Asn Leu Cys Glu 465 470 475 480 Asp Val Asn Gln Gln Phe Val Val Ser Gly Gly Lys Leu Val Gly Ile 485 490 495 Leu Thr Ser Arg Asn Glu Thr Gly Ser Gln Pro Leu Glu Asn Gln Phe 500 505 510 Tyr Ile Lys Ile Thr Asn Gly Thr His Arg Ser Arg Arg Ser Val Asn 515 520 525 Glu Asn Val Thr Asn Cys Pro Tyr Val Ser Tyr Gly Lys Phe Cys Ile 530 535 540 Lys Pro Asp Gly Ser Val Ser Pro Ile Val Pro Lys Glu Leu Glu Gln 545 550 555 560 Phe Val Ala Pro Leu Leu Asn Val Thr Glu Asn Val Leu Ile Pro Asn 565 570 575 Ser Phe Asn Leu Thr Val Thr Asp Glu Tyr Ile Gln Thr His Met Asp 580 585 590 Lys Ile Gln Ile Asn Cys Leu Gln Tyr Val Cys Gly Asn Ser Leu Ala 595 600 605 Cys Arg Lys Leu Phe Gln Gln Tyr Gly Pro Val Cys Asp Asn Ile Leu 610 615 620 Ser Val Val Asn Ser Val Gly Gln Lys Glu Asp Met Glu Leu Leu Ser 625 630 635 640 Phe Tyr Ser Ser Thr Lys Pro Ser Gly Phe Asn Thr Pro Val Phe Ser 645 650 655 Asn Leu Ser Thr Gly Glu Phe Asn Ile Ser Leu Leu Leu Thr Thr Pro 660 665 670 Ser Ser Pro Arg Gly Arg Ser Phe Ile Glu Asp Leu Leu Phe Thr Ser 675 680 685 Val Glu Ser Val Gly Leu Pro Thr Asp Glu Ala Tyr Lys Lys Cys Thr 690 695 700 Ala Gly Pro Leu Gly Phe Leu Lys Asp Leu Ala Cys Ala Arg Glu Tyr 705 710 715 720 Asn Gly Leu Leu Val Leu Pro Pro Ile Ile Thr Ala Glu Met Gln Thr 725 730 735 Leu Tyr Thr Ser Ser Leu Val Val Ser Met Ala Phe Gly Gly Ile Thr 740 745 750 Ser Ala Gly Ala Ile Pro Phe Ala Thr Gln Leu Gln Ala Arg Ile Asn 755 760 765 His Leu Gly Ile Thr Gln Ser Leu Leu Leu Lys Asn Gln Glu Lys Ile 770 775 780 Ala Ala Ser Phe Asn Lys Ala Ile Gly His Met Gln Glu Gly Phe Arg 785 790 795 800 Ser Thr Ser Leu Ala Leu Gln Gln Ile Gln Asp Val Val Asn Lys Gln 805 810 815 Ser Ala Ile Leu Thr Glu Thr Met Ala Ser Leu Asn Lys Asn Phe Gly 820 825 830 Ala Ile Ser Ser Val Ile Gln Glu Ile Tyr Gln Gln Leu Asp Ala Ile 835 840 845 Gln Ala Asn Ala Gln Val Asp Arg Leu Ile Thr Gly Arg Leu Ser Ser 850 855 860 Leu Ser Val Leu Ala Ser Ala Lys Gln Ala Glu Tyr Ile Arg Val Ser 865 870 875 880 Gln Gln Arg Glu Leu Ala Thr Gln Lys Ile Asn Glu Cys Val Lys Ser 885 890 895 Gln Ser Ile Arg Tyr Ser Phe Cys Gly Asn Gly Arg His Val Leu Thr 900 905 910 Ile Pro Gln Asn Ala Pro Asn Gly Ile Val Phe Ile His Phe Ser Tyr 915 920 925 Thr Pro Asp Ser Phe Val Asn Val Thr Ala Ile Val Gly Phe Cys Val 930 935 940 Lys Pro Ala Asn Ala Ser Gln Tyr Ala Ile Val Pro Ala Asn Gly Arg 945 950 955 960 Gly Ile Phe Ile Gln Val Asn Gly Ser Tyr Tyr Ile Thr Ala Arg Asp 965 970 975 Met Tyr Met Pro Arg Ala Ile Thr Ala Gly Asp Ile Val Thr Leu Thr 980 985 990 Ser Cys Gln Ala Asn Tyr Val Ser Val Asn Lys Thr Val Ile Thr Thr 995 1000 1005 Phe Val Asp Asn Asp Asp Phe Asp Phe Asn Asp Glu Leu Ser Lys 1010 1015 1020 Trp Trp Asn Asp Thr Lys His Glu Leu Pro Asp Phe Asp Lys Phe 1025 1030 1035 Asn Tyr Thr Val Pro Ile Leu Asp Ile Asp Ser Glu Ile Asp Arg 1040 1045 1050 Ile Gln Gly Val Ile Gln Gly Leu Asn Asp Ser Leu Ile Asp Leu 1055 1060 1065 Glu Lys Leu Ser Ile Leu Lys Thr Tyr Ile Lys Trp Pro 1070 1075 1080 <210> 13 <211> 1156 <212> PRT <213> Avian infectious bronchitis virus <400> 13 Met Leu Val Lys Ser Leu Phe Ile Val Thr Leu Leu Phe Ala Leu Cys<000249>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 Gln 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> 14 <211> 1078 <212> PRT <213> Avian infectious bronchitis virus <400> 14 Ala Leu Phe Asp Asn Asn Gln Ala Val Tyr Tyr Tyr Gln Ser Ala Phe 1 5 10 15 Arg Pro Ser Ser Gly Trp His Lys His Gly Gly Ala Tyr Ala Val Ala 20 25 30 Asn Val Ser Leu Glu Tyr Ala Asn Ala Gly Ser Ser Thr His Cys Thr 35 40 45 Ala Gly Ala Ile Tyr Trp Ser Lys Asn Phe Thr Ala Ser Ser Val Ala 50 55 60 Met Thr Ala Pro Gly Thr Gly Met Ser Trp Ser Thr Ala Gln Phe Cys 65 70 75 80 Thr Ala His Cys Asn Phe Ser Asp Phe Thr Val Phe Val Thr His Cys 85 90 95 Tyr Lys Ser Gly Asp Val Cys Pro Leu Thr Gly Leu Ile Pro Ser Gly 100 105 110 Tyr Ile Arg Ile Ser Ala Met Thr Lys Gly Thr Thr Ser Leu Phe Tyr 115 120 125 Asn Leu Thr Val Pro Val Thr Lys Tyr Pro Lys Phe Lys Ser Leu Gln 130 135 140 Cys Val Asp Asn Phe Thr Ser Val Tyr Leu Asn Gly Asp Leu Val Phe 145 150 155 160 Thr Ser Asn Glu Thr Lys Asp Val Ser Ala Ala Gly Val His Phe Lys 165 170 175 Ala Gly Gly Pro Ile Thr Tyr Lys Val Met Glu Lys Val Asp Val Leu 180 185 190 Ala Tyr Phe Val Asn Gly Thr Ala Gln Asp Val Ile Leu Cys Asp Asn 195 200 205 Ser Pro Arg Gly Leu Leu Ala Cys Gln Tyr Asn Thr Gly Asn Phe Ser 210 215 220 Asp Gly Phe Tyr Pro Phe Thr Asn Ile Ser Leu Val Lys Glu Lys Phe 225 230 235 240 Ile Val Tyr Arg Glu Thr Ser Val Asn Thr Thr Leu Val Leu Thr Asn 245 250 255 Phe Thr Phe Thr Asn Val Ser Asn Ala Leu Pro Asn Thr Gly Gly Val 260 265 270 Asn Thr Ile Asn Ile Tyr Gln Thr Gln Thr Ala Gln Ser Gly Cys Tyr 275 280 285 Asn Phe Asn Phe Ser Phe Leu Ser Ser Phe Val Tyr Lys Gln Ser Asp 290 295 300 Phe Met Tyr Gly Ser Tyr His Pro Lys Cys Asp Phe Arg Pro Glu Thr 305 310 315 320 Ile Asn Asn Gly Leu Trp Phe Asn Ser Leu Ser Val Ser Leu Ala Tyr 325 330 335 Gly Pro Leu Gln Gly Gly Cys Lys Gln Ser Val Phe Ser Asn Arg Ala 340 345 350 Thr Cys Cys Tyr Ala Tyr Ser Tyr Asn Gly Pro Arg Leu Cys Lys Gly 355 360 365 Val Tyr Ile Gly Glu Leu Gln Gln Tyr Phe Glu Cys Gly Leu Leu Val 370 375 380 Tyr Val Thr Lys Ser Asp Gly Ser Arg Ile Gln Thr Arg Asn Glu Pro 385 390 395 400 Leu Val Leu Thr His His Asn Tyr Asn Asn Ile Thr Leu Asp Arg Cys 405 410 415 Val Glu Tyr Asn Ile Tyr Gly Arg Ser Gly Gln Gly Phe Ile Thr Asn 420 425 430 Val Thr Ala Ala Ala Ala Asn Tyr Asn Tyr Leu Ala Asp Gly Gly Leu 435 440 445 Ala Ile Leu Asp Thr Ser Gly Ala Ile Asp Ile Phe Val Val Gln Gly 450 455 460 Glu Tyr Gly Pro Asn Tyr Tyr Lys Val Asn Pro Cys Glu Asp Val Asn 465 470 475 480 Gln Gln Phe Val Val Ser Gly Gly Gly Ile Val Gly Val Leu Thr Ser 485 490 495 His Asn Glu Thr Gly Ser Gln Gln Leu Glu Asn Leu Phe Tyr Val Lys 500 505 510 Leu Thr Asn Ser Thr Arg Arg Thr Arg Arg Ser Thr Ile Ala Asn Val 515 520 525 Thr Thr Cys Pro Tyr Val Ser Tyr Gly Arg Phe Cys Ile Lys Pro Asp 530 535 540 Gly Leu Val Ser Glu Ile Val Pro Gln Glu Leu Asp Tyr Phe Val Ala 545 550 555 560 Pro Leu Leu Asn Val Thr Glu His Val Leu Ile Pro Asn Ser Phe Asn 565 570 575 Leu Thr Val Thr Asp Glu Tyr Ile Gln Thr Arg Met Glu Lys Val Gln 580 585 590 Ile Asn Cys Leu Gln Tyr Val Cys Gly Asn Ser Ile Glu Cys Arg Asn 595 600 605 Leu Phe Gln Gln Tyr Gly Pro Val Cys Asp Asn Ile Leu Ser Ile Val 610 615 620 Asn Ser Val Gly Gln Arg Glu Asp Met Glu Ser Leu Thr Phe Tyr Ser 625 630 635 640 Ser Thr Lys Pro Lys Gly Tyr Asn Thr Pro Ile Phe Ser Asn Ile Ser 645 650 655 Thr Gly Asp Phe Asn Ile Ser Leu Met Leu Thr Pro Pro Ser Ser Pro 660 665 670 Ser Gly Arg Ser Phe Ile Glu Asp Leu Leu Phe Thr Ser Val Glu Thr 675 680 685 Val Gly Leu Pro Thr Asp Ala Glu Tyr Lys Lys Cys Thr Ala Gly Pro 690 695 700 Leu Gly Thr Leu Lys Asp Leu Ile Cys Ala Arg Glu Tyr Asn Gly Leu 705 710 715 720 Leu Val Leu Pro Pro Ile Ile Thr Ala Asp Met Gln Thr Met Tyr Thr 725 730 735 Ala Ser Leu Val Gly Ala Met Ala Phe Gly Gly Ile Thr Ser Ala Ala 740 745 750 Ala Ile Pro Phe Ala Thr Gln Ile Gln Ala Arg Ile Asn His Leu Gly 755 760 765 Ile Thr Gln Ser Leu Leu Met Lys Asn Gln Glu Lys Ile Ala Ala Ser 770 775 780 Phe Asn Lys Ala Ile Gly His Met Gln Glu Gly Phe Arg Ser Thr Ser 785 790 795 800 Leu Ala Leu Gln Gln Ile Gln Asp Val Val Asn Lys Gln Ser Ala Ile 805 810 815 Leu Thr Glu Thr Met Asn Ser Leu Asn Lys Asn Phe Gly Ala Ile Thr 820 825 830 Ser Val Ile Gln Asp Ile Tyr Ala Gln Leu Asp Ala Ile Gln Ala Asp 835 840 845 Ala Gln Val Asp Arg Leu Ile Thr Gly Arg Leu Ser Ser Leu Ser Val 850 855 860 Leu Ala Ser Ala Lys Gln Ser Glu Tyr Ile Arg Val Ser Gln Gln Arg 865 870 875 880 Glu Leu Ala Thr Gln Lys Ile Asn Glu Cys Val Lys Ser Gln Ser Asn 885 890 895 Arg Tyr Gly Phe Cys Gly Ser Gly Arg His Val Leu Ser Ile Pro Gln 900 905 910 Asn Ala Pro Asn Gly Ile Val Phe Ile His Phe Thr Tyr Thr Pro Glu 915 920 925 Ser Phe Val Asn Val Thr Ala Ile Val Gly Phe Cys Val Ser Pro Ala 930 935 940 Asn Ala Ser Gln Tyr Ala Ile Val Pro Ala Asn Gly Arg Gly Ile Phe 945 950 955 960 Ile Gln Val Asn Gly Thr Tyr Tyr Ile Thr Ala Arg Asp Met Tyr Met 965 970 975 Pro Arg Asp Ile Thr Ala Gly Asp Ile Val Thr Leu Thr Ser Cys Gln 980 985 990 Ala Asn Tyr Val Asn Val Asn Lys Thr Val Ile Thr Thr Phe Val Glu 995 1000 1005 Asp Asp Asp Phe Asp Phe Asp Asp Glu Leu Ser Lys Trp Trp Asn 1010 1015 1020 Glu Thr Lys His Glu Ile Pro Asp Phe Asp Glu Phe Asn Tyr Thr 1025 1030 1035 Val Pro Ile Leu Asn Ile Ser Ser Glu Ile Asp Arg Ile Gln Gly 1040 1045 1050 Val Ile Gln Gly Leu Asn Asp Ser Leu Ile Asn Leu Glu Glu Leu 1055 1060 1065 Ser Ile Ile Lys Thr Tyr Ile Lys Trp Pro 1070 1075 <210> 15 <211> 1169 <212> PRT <213> Avian infectious bronchitis virus <400> 15 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 Gln 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> 16 <211> 1082 <212> PRT <213> Avian infectious bronchitis virus <400> 16 Ser Leu Tyr Asn Asn Asp Ser Tyr Val Tyr Tyr Tyr Gln Ser Ala Phe 1 5 10 15 Arg Pro Phe Asn Gly Trp His Leu His Gly Gly Ala Tyr Ala Val Val 20 25 30 Asn Val Ser Gln Glu Thr Ala Asn Ala Gly Ser Ser Pro Ser Cys Thr 35 40 45 Ala Gly Ala Ile Tyr Trp Ser Lys Asn Phe Thr Ala Ser Ser Val Ala 50 55 60 Met Thr Ala Pro Leu Gln Gly Met Gln Trp Ser Thr Ile Gln Phe Cys 65 70 75 80 Thr Ala His Cys Asn Phe Thr Asn Ile Val Val Phe Val Thr His Cys 85 90 95 Tyr Lys Ser Gly Ser Thr Val Cys Pro Leu Thr Gly Leu Ile Pro Gln 100 105 110 Asn His Ile Arg Ile Ser Ala Met Lys Gln Gly Asn Asn Gly Pro Ser 115 120 125 Gly Leu Phe Tyr Asn Leu Thr Val Pro Val Thr Lys Tyr Ser Lys Phe 130 135 140 Lys Ser Leu Gln Cys Val Asn Asn Gln Thr Ser Val Tyr Leu Asn Gly 145 150 155 160 Asp Leu Val Phe Thr Ser Asn Glu Thr Lys Asp Val Ser Gly Ala Gly 165 170 175 Val Tyr Phe Lys Ala Gly Gly Pro Ile Thr Tyr Lys Val Met Arg Glu 180 185 190 Val Lys Ala Leu Ala Tyr Phe Val Asn Gly Thr Ala His Asp Val Ile 195 200 205 Leu Cys Asp Gly Ser Pro Arg Gly Leu Leu Ala Cys Gln Tyr Asn Thr 210 215 220 Gly Lys Phe Ser Asp Gly Phe Tyr Pro Phe Thr Asn Asp Thr Leu Val 225 230 235 240 Lys Glu Lys Phe Ile Val Tyr Arg Glu Asn Ser Val Asn Thr Thr Leu 245 250 255 Thr Leu Thr Asn Phe Thr Phe Tyr Asn Glu Ser Asn Ala Leu Pro Asn 260 265 270 Asn Gly Gly Val Asp Thr Ile Gln Leu Tyr Gln Thr His Thr Ala Gln 275 280 285 Ser Gly Tyr Tyr Asn Phe Asn Phe Ser Phe Leu Ser Ser Phe Gln Tyr 290 295 300 Val Glu Ser Asn Phe Met Tyr Gly Ser Tyr His Pro Lys Cys Gly Phe 305 310 315 320 Arg Pro Glu Ser Ile Asn Asn Gly Leu Trp Phe Asn Ser Leu Ser Val 325 330 335 Ser Leu Ala Tyr Gly Pro Leu Gln Gly Gly Cys Lys Gln Ser Val Phe 340 345 350 His Gly Arg Ala Thr Cys Cys Tyr Ala Tyr Ser Tyr Asn Gly Pro Thr 355 360 365 Leu Cys Lys Gly Val Tyr Ser Gly Glu Leu Thr Arg Ser Tyr Gln Cys 370 375 380 Gly Leu Leu Val Phe Val Thr Lys Ser Asp Gly Ser Arg Ile Gln Thr 385 390 395 400 Ser Thr Lys Pro Ile Val Leu Thr Gln His Asn Tyr Asn Asn Ile Thr 405 410 415 Leu Asp Arg Cys Val Glu Tyr Asn Ile Tyr Gly Arg Val Gly Gln Gly 420 425 430 Phe Ile Thr Asn Val Thr Glu Ser Ala Ala Ala Phe Asn Tyr Leu Glu 435 440 445 Asp Gly Gly Leu Ala Ile Leu Asp Thr Ser Gly Ala Ile Asp Ile Phe 450 455 460 Val Val Gln Gly Glu Tyr Gly Phe Asn Tyr Tyr Lys Val Asn Pro Cys 465 470 475 480 Glu Asp Val Asn Gln Gln Phe Val Val Ser Gly Gly Asn Leu Val Gly 485 490 495 Ile Leu Thr Ser Ile Asn Gln Thr Gly Ser Gln Ser Ile Glu Asn Gln 500 505 510 Phe Tyr Val Lys Leu Thr Asn Gly Ser Arg Arg Ser Arg Arg Ser Val 515 520 525 Ser Glu Asn Val Thr Ser Cys Pro Tyr Val Ser Tyr Gly Lys Phe Cys 530 535 540 Ile Lys Pro Asp Gly Ser Leu Ser Thr Ile Val Pro Lys Glu Leu Glu 545 550 555 560 Gln Phe Val Ala Pro Leu Leu Asn Val Thr Glu His Val Leu Ile Pro 565 570 575 Asp Ser Phe Asn Leu Thr Val Thr Asp Glu Tyr Ile Gln Thr Arg Met 580 585 590 Asp Lys Val Gln Ile Asn Cys Leu Gln Tyr Val Cys Gly Asn Ser Phe 595 600 605 Glu Cys Arg Lys Leu Phe Gln Gln Tyr Gly Pro Val Cys Asp Asn Ile 610 615 620 Leu Ser Val Val Asn Ser Val Gly Gln Lys Glu Asp Met Glu Leu Leu 625 630 635 640 Ser Phe Tyr Ser Ser Thr Lys Pro Ser Gly Ile Ser Gln Pro Leu Phe 645 650 655 Asn Asn Phe Ser Thr Gly Asp Phe Asn Ile Ser Leu Leu Leu Thr Ser 660 665 670 Pro Ser Ser Pro Ser Gly Arg Ser Phe Ile Glu Asp Leu Leu Phe Thr 675 680 685 Ser Val Glu Ser Val Gly Leu Pro Thr Asp Glu Ala Tyr Lys Lys Cys 690 695 700 Thr Ser Gly Pro Leu Gly Phe Val Lys Asp Leu Val Cys Ala Arg Glu 705 710 715 720 Tyr Asn Gly Leu Leu Val Leu Pro Pro Ile Ile Thr Ala Glu Met Gln 725 730 735 Thr Met Tyr Thr Ser Ser Leu Val Ala Ser Met Ala Leu Gly Gly Ile 740 745 750 Thr Ala Ala Gly Ala Ile Pro Phe Ala Thr Gln Leu Gln Ala Arg Ile 755 760 765 Asn His Leu Gly Ile Thr Gln Ser Leu Leu Met Lys Asn Gln Glu Lys 770 775 780 Ile Ala Ala Ser Phe Asn Arg Ala Ile Gly His Met Gln Glu Gly Phe 785 790 795 800 Lys Ser Thr Ser Leu Ala Leu Gln Gln Ile Gln Asp Val Val Asn Lys 805 810 815 Gln Ser Ala Ile Leu Thr Glu Thr Met Ala Ser Leu Asn Lys Asn Phe 820 825 830 Gly Ala Ile Ser Ser Val Leu Gln Asp Ile Tyr Gln Gln Leu Asp Val 835 840 845 Ile Gln Ala Asp Ala Gln Val Asp Arg Ile Ile Thr Gly Arg Leu Ser 850 855 860 Ser Leu Ser Val Leu Ala Ser Ala Lys Gln Ser Glu Tyr Ile Ala Val 865 870 875 880 Ser Gln Gln Arg Ala Leu Ala Thr Gln Lys Ile Asn Glu Cys Val Lys 885 890 895 Ser Gln Ser Thr Arg Tyr Ser Phe Cys Gly Asn Gly Arg His Val Leu 900 905 910 Thr Ile Pro Gln Asn Ala Pro Asn Gly Ile Val Phe Ile His Phe Thr 915 920 925 Tyr Thr Pro Glu Ser Phe Val Asn Val Thr Ala Ile Val Gly Phe Cys 930 935 940 Val Lys Pro Pro Asn Ala Ser His Tyr Ala Ile Val Pro Val Asn Gly 945 950 955 960 Arg Gly Ile Phe Ile Gln Val Asn Gly Thr Tyr Tyr Ile Thr Ser Arg 965 970 975 Asp Met Tyr Met Pro Arg Asn Ile Thr Ala Gly Asp Ile Val Thr Leu 980 985 990 Thr Ser Cys Gln Ala Asn Tyr Val Ser Val Asn Lys Thr Val Ile Ser 995 1000 1005 Thr Phe Val Glu Asp Asp Asp Phe Asp Phe Asp Asp Glu Leu Ser 1010 1015 1020 Lys Trp Trp Asn Asp Thr Lys His Glu Leu Pro Asp Phe Asp Glu 1025 1030 1035 Phe Asn Tyr Thr Ile Pro Val Leu Asn Ile Ser Asn Glu Ile Asp 1040 1045 1050 Arg Ile Gln Gly Val Ile Gln Gly Leu Asn Asp Ser Leu Ile Asp 1055 1060 1065 Leu Glu Thr Leu Ser Ile Leu Lys Thr Tyr Ile Lys Trp Pro 1070 1075 1080 <210> 17 <211> 3495 <212> DNA <213> Avian infectious bronchitis virus <400> 17 atgttggaca aaccgctttt actagtgact ctttggtatg cactatgtag tgctttgctc 60 tatgataata atacttacgt ttactactac caaagtgcct ttaggcctgg tccaggttgg 120 cacctatatg ggggtgctta tgcagtagat agggttttta atgaaaccaa caatgcaggc 180 agtgcatctg attgcactgc tggtactttt tatgaaagcc ataatatttc tgcttcttct 240 gtagccatga cagtaccaca taatggtatg tcttggtcag cttcacaatt ttgtacagct 300 cattgtaact tctcagactt tacagtgttc gttacgcatt gttttaaaaa tcaactcggt 360 agttgtccct tgacaggtat gattcctcag aatcatattc gtatttctgc tatgagagat 420 ggagttttgt tttataactt aacagttagc gtatctaaat accctagatt taaatcgctt 480 caatgtgtta gcaattctac atctgtctat gtaaatggtg accttgtttt cacttctaat 540 gaaacttctt acgttacggg tgcaggcgtt tattttaaaa gtggtgggcc tgtaacttat 600 aaagttatga aagaagttaa agccctagcc tactttatta atggtaccgc acaagaggtt 660 atttatgtg ataactcacc tagaggttg cttgcatgtc agtataacac tggtaatttt 720 tcagatggat tctacccttt tactaatcat tctttagtta aggataggtt tattgtatat 780 cgagaaagta gcactaacac tactttaaag ttaactaatt tcagttttac taatgtaagt 840 aatgcttctc ctaattcagg tggcgttgat actttccaat tatatcaaac aagtactgct 900 caggatggtt attataattt taatttatca tttctgagta gttttgtgta taaaccatct 960 gatttatgt atgggtcata ccacccacat tgtaagttta gaccagagaa tattaataat 1020 ggcttatggt ttaattcatt atctgtgtca cttacttacg gacccattca aggtggttgt 1080 aagcaatctg tttttagtaa tagagcaact tgttgctatg cttattctta tcaagggcct 1140 agtagatgta agggtgttta tagaggggag ctaacgcaat actttgaatg tggacttcta 1200 gttacgtaa ctaagagtga tggctctcgt atacaaacta gaagtgaacc actggtgtta 1260 actcaata attataaca cattacttta ataagtgtg ttgagtataa tatatatggt 1320 agggttggtc aaggttttat tactaatgta actgaagcaa ctgctatta tagttatcta 1380 gcagatgtg gtttagctat tttagatacc tcaggagcca tagacatatt tgttgttcaa 1440 gtcttatta ttataggtt aatccctgtg aagatgttaa ccaacagttt 1500 gtgtgtctg gtggcaactt cttacatctc ataatgaac aggttctgaa 1560 tctattgaga accagtttta catcaactc actaacggaa cacgtcgctc tagacgttct 1620 gttactggga atgttacaa ttgccctt gttagttatg gcaagttttg tataaaacca 1680 gatggttctt tatctataat agtaccacaa gattagaac agtttgtggc gccttttattc 1740 aatgttactg agcatgtgct catacctgat agttttaatt taactgtcac agatgagtac 1800 attackaactc gtatggataa gttcaatt atttgccttc agtatgtttg tggtattct 1860 attgaatgca gaaagttgtt tcagcagtat ggacctgttt gtgatatatat attgtctgtt 1920 gtaaatggtg taggtcaag agaggatatg gaacttttaa gttctattc ttctaaa 1980 cctagtggtt acaatacacc aatttttaat aatgttagca ctggtgactt tatattcg 2040 ctcctactaa caccacctaa tagtcctact gggcgctctt ttattgaaga tcttctcttt 2100 acaagtgtag aatctgttgg attaccaact gatgaagagt ataaaaagtg tacagcagga 2160 cctttaggtt ttgttaaaga ccttgtttgt gctagagagt ataatggtttt gctcgttctg 2220 cctcctatta ttactgcgga aatgcaaacc atgtatacta gttctttagt agcctctatg 2280 gctttaggtg gcattactgc agctggtgct ataccttttg ctacacaact gcaggccaga 2340 attaaccatt tgggtattac taattctctt ttgttgaaaa accaagaaaa aattgctgct 2400 tccttaata aggccatcgg tcatatgcag gaagggttta aaagtacttc tctagcatta 2460 caacagattc aagatgttgt tataaacag agttctattc ttacagagac tatgcaatca 2520 cttaataaaa atttggtgc tatttcctct gtaattcaag acatttacca gcaactagat 2580 gctattcagg cagatgctca ggttgatcgt cttattacag gtagactctc ttcactatct 2640 gttttagctt ctgctaaaca ggcagagtat catagagtgt cacaacagcg tgagttggcc 2700 actcagaaaa ttaatgagtg tgttaagtct cagtctaata ggtattcatt ttgtggtaat 2760 ggtagacatg ttctaaccat accacagaat gcacccaatg gcatagtgtt tatacacttt 2820 acatacactc cagagagttt tgttaatgtt acggcaatag tagggttttg cgtaaaccca 2880 gctaatgcta gtcattatgc aatagtgcct gttaatggca ggggtgtttt tatagaagtt 2940 aatggtagtt actatatcac tgctcgtgat atgtatatgc caagagatat tactgcagga 3000 gacatagtca ctttgacttc ttgtcaagca aactatgtta atgtaaataa aaccgtcatt 3060 aacactttg tggaagatga cgattttgat tttatgatg aattgtcaaa atggtggaat 3120 gatactaagc atgagctacc agattttgat gaattcaatt ataccgttcc agttttaaat 3180 attagtaatg aaattgacag aattcaacag gttattcagg gattaaatga ttccctaata 3240 gaccttgaaa cactctcaat tctcaaaact tatattaaat ggccttggta tgtgtggctt 3300 gccattgcat tccttaccat tatttttatt ctggtacttt gttggatatt tttcatgacc 3360 ggttgttgcg gttgttgttg tggatgcttt ggtatcatac cgttaatgag taagtgtggt 3420 aagaaatctt cttactacac gacttttgat aatgatgtgg taacttaaca atacagacct 3480 aaaaagtctg tttaa 3495 <210> 18 <211> 3225 <212> DNA <213> Avian infectious bronchitis virus <400> 18 gctttgtatg actcgagttc ttacgtgtac tactaccaaa gtgccttcag accacctgat 60 ggttggcatt tacatggggg tgcgtatgcg gttgttaata tttctagtga atctaataat 120 gcaggctctt catctgggtg tactgttggt attattcatg gtggtcgtgt tgttaatgct 180 tcttctatag ctatgacggc accgtcatca ggtatggctt ggtctagcag tcagttttgt 240 actgcatact gtaacttttc agatactaca gtgtttgtta cacattgtta taaacatggt 300 gggtgtccta taactggcat gcttcaacag cattctatac gtgtttctgc tatgaaaaat 360 ggccagcttt tctataattt aacagttagt gtagctaagt accctacttt taaatcattt 420 cagtgtgtta ataatttaac atccgtatat ttaaatggtg atcttgttta cacctctaat 480 gagaccacag atgttacatc tgcaggtgtt tattttaaag ctggtggacc tataacttat 540 aaagttatga gagaagttag agccctggct tattttgtta atggtactgc acaagatgtt 600 atttgtgtg atgggtcacc tagaggcttg ttagcatgcc agtataac tggcaatttt 660 tcagatggct ttatccttt tactaatagt agtttagtta agcagaagtt tattgtctat 720 cgtgaaaata gtgttaatac tactttacg ttacacatt tcactttca taatgagact 780 ggcgccaacc aaatcctag tggtgtccag atattcaa cctaccaac acaacagct 840 cagagtggtt attaattt taatttttcc tttctgagta gttttgttta taaggagtct 900 aattttatgt atggatctta tcaccagt tgtatttta gactagaac tattaataat 960 ggtttgtggt ttaattcact ttcagttagt attgctcg gtcctctca aggtggttgc 1020 aagcaatctg tctttagtgg tagagcacc tgttgttg cttactcata tggaggtcct 1080 ttgctgtgta aaggtgttta ttcaggtgag ttagatcata attttgaatg tggactgtta 1140 gtttatgtta ctaagagcgg tggctctcgt atacaacag ccactgaacc gccagttata 1200 actcacaca attatata tattacttta atacttgtg ttgattataa tatatatggc 1260 agaactggcc agggttttat tactaatgta accgactcag ctgttagtta taattatcta 1320 gcagacgcag gtttggctat tttagataca tctggttcca tagacatctt tgtcgtacaa 1380 agtgaatatg gtcttaatta ttataaggtt aacccttgcg aagatgtcaa ccagcagttt 1440 gtagttctg gtggtaaatt agtaggtatt cttacttcac gtaatgagac tggttcccag 1500 cttcttgaga atcagtttta catcaaaatc actaatggaa cacgtcgttt tagacgttct 1560 attactgaaa gtgttgaaaa ttgcccttat gttagttatg gtaagttttg tataaaacct 1620 gatggcagta ttgccacaat agtaccaaaa cagttagaac agtttgtggc acctttactt 1680 aatgttactg aaaatgtgct catacctaac agttttaatt taactgttac agatgagtac 1740 atacaaactc ggatggataa ggtccaaatt aattgcctgc agtatatttg tggcaattct 1800 ctggagtgca gaaatttgtt tcaacaatat ggtcctgttt gcgacaacat attgtctgta 1860 gtaaatagtg ttggtcaaaa agaagatatg gaacttttga atttctattc ttctactaag 1920 ccggctggtt ttaatacacc agttcttagt aatgttagca ctggtgagtt tatattact 1980 cttttttaa caacgcctag tagtcctaga aggcgttctt ttattgaaga ccttctattt 2040 acaagtgttg aatctgttgg attaccaaca gatgacgcat acaaaaattg cactgcaggt 2100 cctttaggct ttctgaaaga cttgcatgt gctcgtgaat ataatggtttt gcttgtgttg 2160 cctcctatta taacagcaga aatgcaaact ttgtatacaa gctctctagt agcttctatg 2220 gcttttggtg gtattactgc agctggtgct ataccttttg ccacacaact gcaggctaga 2280 attaatcact tgggtattac ccagtcactt cttttgaaga atcaagaaaa aattgctgct 2340 tccttaata aggccatcgg tcatatgcag gaaggtttta gaagtacatc tttagcatta 2400 caacaaattc aagatgttgt tataagcag agtgctattc ttactgagac tatggcatca 2460 cttaataaaa atttttgtgc catttcttct gtgattcaag aaatctacca gcaacttgac 2520 gccatacaag caaatgctca agtggatcgt cttataactg gtagattgtc atcactttct 2580 gtttagcat ctgctaagca ggcggagtat attagagtgt cacaacagcg tgagttagct 2640 actcagaaga ttaatgagtg tgttaagtca cagtccatta ggtactcctt ttgtggtaat 2700 ggacgacatg ttttaaccat accgcaaaat gcacctaatg gtatagtgtt tatacacttt 2760 tcttacactc cagatagttt tgttaatgtt actgcaatag tgggtttttg tgtaaagcca 2820 gctaatgcta gtcagtatgc aatagtaccc gctaatggta ggggtatttt tatacaagtt 2880 aatggtagtt actacatcac tgcacgagat atgtatatgc caagagctat tactgcagga 2940 gatatagtta cgcttacttc ttgtcaagca aattatgtaa gtgtaaataa gaccgtcatt 3000 actacattcg tagacaatga tgattttgat tttaatgacg aattgtcaaa atggtggaat 3060 gatactaagc atgagctacc agactttgac aaattcaatt acacagtacc tatacttgac 3120 attgatagtg aaattgatcg tattcaaggc gttatacagg gtcttaatga ctctctaata 3180 gaccttgaaa aactttcaat actcaaaact tatattaagt ggcct 3225 <210> 19 <211> 11398 <212> DNA <213> Artificial Sequence <220> <223> Plasmid Sequence with IBV <400> 19 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 ggagaaagt 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> 20 <211> 3231 <212> DNA <213> Avian infectious bronchitis virus <400> 20 ttgctctatg ataataatac ttacgtttac tactaccaaa gtgcctttag gcctggtcca 60 ggttggcacc tatatggggg tgcttatgca gtagataggg tttttaatga aaccaacaat 120 gcaggcagtg catctgattg cactgctggt actttttatg aaagccataa tatttctgct 180 tcttctgtag ccatgacagt accacataat ggtatgtctt ggtcagcttc acaattttgt 240 acagctcatt gtaacttctc agactttaca gtgttcgtta cgcattgttt taaaaatcaa 300 ctcggtagtt gtcccttgac aggtatgatt cctcagaatc atattcgtat ttctgctatg 360 agagatggag ttttgtttta taacttaaca gttagcgtat ctaaataccc tagatttaaa 420 tcgcttcaat gtgttagcaa ttctacatct gtctatgtaa atggtgacct tgttttcact 480 tctaatgaaa cttcttacgt tacgggtgca ggcgtttatt ttaaaagtgg tgggcctgta 540 acttataaag ttatgaaaga agttaaagcc ctagcctact ttattaatgg taccgcacaa 600 gaggttattt tatgtgataa ctcacctaga ggtttgcttg catgtcagta taacactggt 660 aatttttcag atggattcta cccttttact aatcattctt tagttaagga taggtttatt 720 gtatatcgag aaagtagcac taacactact ttaaagttaa ctaatttcag ttttactaat 780 gtaagtaatg cttctcctaa ttcaggtggc gttgatactt tccaattata tcaaacaagt 840 actgctcagg atggttatta taattttaat ttatcatttc tgagtagttt tgtgtataaa 900 ccatctgatt ttatgtatgg gtcataccac ccacattgta agtttagacc agagaatatt 960 aataatggct tatggtttaa ttcattatct gtgtcactta cttacggacc cattcaaggt 1020 ggttgtaagc aatctgtttt tagtaataga gcaacttgtt gctatgctta ttcttatcaa 1080 gggcctagta gatgtaaggg tgtttataga ggggagctaa cgcaatactt tgaatgtgga 1140 cttctagttt acgtaactaa gagtgatggc tctcgtatac aaactagaag tgaaccactg 1200 gtgttaactc aataatatta taacaacatt actttaaata agtgtgttga gtataatata 1260 tatggtaggg ttggtcaagg ttttattact aatgtaactg aagcaactgc taattatagt 1320 tatctagcag atggtggttt agctatttta gatacctcag gagccataga catatttgtt 1380 gttcaaggtg catatggtct taattattat aaggttaatc cctgtgaaga tgttaaccaa 1440 cagtttgtag tgtctggtgg caacttagtt ggcattctta catctcataa tgaaacaggt 1500 tctgaatcta ttgagaacca gttttacatc aaactcacta acggaacacg tcgctctaga 1560 cgttctgtta ctgggaatgt tacaaattgc ccttatgtta gttatggcaa gttttgtata 1620 aaaccagatg gttctttatc tataatagta ccacaagaat tagaacagtt tgtggcgcct 1680 ttattcaatg ttactgagca tgtgctcata cctgatagtt ttaatttaac tgtcacagat 1740 gagtacatac aaactcgtat ggataaggtt caaattattt gccttcagta tgtttgtggt 1800 aattctattg aatgcagaaa gttgtttcag cagtatggac ctgtttgtga tatatattg 1860 tctgttgtaa atggtgtagg tcaaagagag gatatggaac ttttaagtttt ctattcttct 1920 actaaaccta gtggttacaa tacaccaatt tttaataatg ttagcactgg tgactttaat 1980 atttcgctcc tactaacacc acctaatagt cctactgggc gctcttttat tgaagatctt 2040 ctctttacaa gtgtagaatc tgttggatta ccaactgatg aagagtataa aaagtgtaca 2100 gcaggacctt taggttttgt taaagacctt gtttgtgcta gagagtataa tggtttgctc 2160 gttctgcctc ctattattac tgcggaaatg caaaccatgt atactagttc tttagtagcc 2220 tctatggctt taggtggcat tactgcagct ggtgctatac cttttgctac acaactgcag 2280 gccagaatta accatttggg tattactaat tctctttttgt tgaaaaacca agaaaaaatt 2340 gctgcttcct ttaataaggc catcggtcat atgcaggaag ggttaaaag tacttctcta 2400 gcattacaac agattcaaga tgttgttaat aaacagagtt ctattcttac agagactatg 2460 caatcactta ataaaaattt tggtgctatt tcctctgtaa ttcaagacat ttaccagcaa 2520 ctagatgcta ttcaggcaga tgctcaggtt gatcgtctta ttacaggtag actctcttca 2580 ctatctgttt tagcttctgc taaacaggca gagtatcata gagtgtcaca acagcgtgag 2640 ttggccactc agaaaattaa tgagtgtgtt aagtctcagt ctaataggta ttcatttgtgt 2700 ggtaatggta gacatgttct aaccatacca cagaatgcac ccaatggcat agtgtttata 2760 cactttacat acactccaga gagttttgtt aatgttacgg caatagtagg gttttgcgta 2820 aacccagcta atgctagtca ttatgcaata gtgcctgtta atggcagggg tgtttttata 2880 gaagttaatg gtagttacta tatcactgct cgtgatatgt atatgccaag agatattact 2940 gcaggagaca tagtcacttt gacttcttgt caagcaaact atgttaatgt aaataaaacc 3000 gtcattaaca cttttgtgga agatgacgat tttgattttt atgatgaatt gtcaaaatgg 3060 tggaatgata ctaagcatga gctaccagat tttgatgaat tcaattatac cgttccagtt 3120 ttaaatatta gtaatgaaat tgacagaatt caacaggtta ttcagggatt aaatgattcc 3180 ctaatagacc ttgaaacact ctcaattctc aaaacttata ttaaatggcc t 3231 <210> 21 <211> 11404 <212> DNA <213> Artificial sequence <220> <223> Plasmid with IBV <400> 21 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 catacctcta agggcttttg 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 agtgctttgc tctatgataa tatacttac 1740 gtttactact accaaagtgc ctttaggcct ggtccaggtt ggcacctata tgggggtgct 1800 tatgcagtag atagggttt taatgaaacc aacaatgcag gcagtgcatc tgattgcact 1860 gctggtactt tttatgaaag ccataatatt tctgcttctt ctgtagccat gacagtacca 1920 1980 tttacagtgt tcgttacgca ttgttttaaa aatcaactcg gtagttgtcc cttgacaggt 2040 atgattcctc agaatcatat tcgtatttct gctatgagag atggagtttt gttttataac 2100 ttaacagtta gcgtatctaa ataccctaga tttaaatcgc ttcaatgtgt tagcaattct 2160 acatctgtct atgtaaatgg tgaccttgtt ttcacttcta atgaaacttc ttacgttacg 2220 ggtgcaggcg tttattttaa aagtggtggg cctgtaactt ataaagttat gaaagaagtt 2280 aaagccctag cctactttat taatggtacc gcacaagagg ttattttatg tgataactca 2340 cctagaggtt tgcttgcatg tcagtataac actggtaatt tttcagatgg attctaccct 2400 tttactaatc attctttagt tagggatagg tttattgtat atcgagaaag tagcactaac 2460 actactttaa agttaactaa tttcagtttt actaatgtaa gtaatgcttc tcctaattca 2520 ggtggcgttg atactttcca attatatcaa acaagtactg ctcaggatgg ttattataat 2580 tttaatttat catttctgag tagttttgtg tataaaccat ctgattttat gtatgggtca 2640 taccacccac attgtaagtt tagaccagag aatattaata atggcttatg gtttaattca 2700 ttatctgtgt cacttactta cggacccatt caaggtggtt gtaagcaatc tgtttttagt 2760 aatagagcaa cttgttgcta tgcttattct tatcaagggc ctagtagatg taagggtgtt 2820 tatagagggg agctaacgca atactttgaa tgtggacttc tagtttacgt aactaagagt 2880 gatggctctc gtatacaaac tagaagtgaa ccactggtgt taactcaata taattataac 2940 aacattactt taaataagtg tgttgagtat aatatatatg gtagggttgg tcaaggtttt 3000 attactaatg taactgaagc aactgctaat tatagttatc tagcagatgg tggtttagct 3060 atttagata cctcaggagc catagacata tttgttgttc aaggtgcata tggtcttaat 3120 tattataagg ttaatccctg tgaagatgtt aaccaacagt ttgtagtgtc tggtggcaac 3180 ttagttggca ttcttacatc tcataatgaa acaggttctg aatctattga gaaccagttt 3240 tacatcaaac tcactaacgg aacacgtcgc tctagacgtt ctgttactgg gaatgttaca 3300 aattgccctt atgttagtta tggcaagttt tgtataaaac cagatggttc tttatctata 3360 atagtaccac aagaattaga acagtttgtg gcgcctttat tcaatgttac tgagcatgtg 3420 ctcatacctg atagttttaa tttaactgtc acagatgagt acatacaaac tcgtatggat 3480 aaggttcaaa ttattgcct tcagtatgtt tgtggtaatt ctattgaatg cagaaagttg 3540 tttcagcagt atggacctgt ttgtgataat atattgtctg ttgtaaatgg tttaggtcaa 3600 agagaggata tggaactttt aagtttctat tcttctacta aacctagtgg ttacaataca 3660 ccaattttta ataatgttag cactggtgac tttaatattt cgctcctact aacaccacct 3720 aatagtccta ctgggcgctc ttttattgaa gatcttctct ttacaagtgt agaatctgtt 3780 ggattaccaa ctgatgaaga gtataaaaaag tgtacagcag gacctttagg ttttgttaaa 3840 gaccttgttt gtgctagaga gtataatggt ttgctcgttc tgcctcctat tattactgcg 3900 gaaatgcaaa ccatgtatac tagttcttta gtagcctcta tggctttagg tggcattact 3960 gcagctggtg ctataccttt tgctacacaa ctgcaggcca gaattaacca tttgggtatt 4020 actaattctc ttttgttgaa aaaccaagaa aaaattgctg cttcctttaa taaggccatc 4080 ggtcatatgc aggaagggtt taaaagtact tctctagcat tacaacagat tcaagatgtt 4140 gttaataaac agagttctat tcttacagag actatgcaat cacttaataa aaatttggt 4200 gctatttcct ctgtaattca agacatttac cagcaactag atgctattca ggcagatgct 4260 caggttgatc gtcttattac aggtagactc tcttcactat ctgttttagc ttctgctaaa 4320 caggcagagt atcatagagt gtcacaacag cgtgagttgg ccactcagaa aattaatgag 4380 tgtgttaagt ctcagtctaa taggtattca ttttgtggta atggtagaca tgttctaacc 4440 ataccacaga atgcacccaa tggcatagtg tttatacact ttacatacac tccagagagt 4500 tttgttaatg ttacggcaat agtagggttt tgcgtaaacc cagctaatgc tagtcattat 4560 gcaatagtgc ctgttaatgg caggggtgtt tttatagaag ttaatggtag ttactatatc 4620 actgctcgtg atatgtatat gccaagagat attactgcag gagacatagt cactttgact 4680 tcttgtcaag caactatgt taatgtaat aaaccgtca tcaactt tgtggagat 4740 gacgattttg atttttatga tgaattgtca aaatggtgga atgatactaa gcatgagcta 4800 ccagattttg atgaattcaa ttataccgtt ccagttttaa atattagtaa tgaaattgac 4860 agaattcaac aggttattca gggattaat gattccctaa tagaccttga aacactctca 4920 attctcaaaa cttatattaa atggccttgg tatgtgtggc tagccatagc tttgccact 4980 attatcttca tcttaatatt aggatggtt ttctcatga ctggttgttg tggttgttgtt 5040 tgtggatgct ttggcattat gcctctaatg agtaagtgtg gtaagaaatc ttcttattac 5100 acgactttg ataacgatgt ggtaactgaa caatacagac ctaaaaagtc tgtttaatga 5160 tccaaagtcc cactagtttc ttaatagtat taatttgct ttggtgtaaa cttgtactaa 5220 gttgttttag agagtttatt attgccctc aacaactaac acagtttta ctccaaatta 5280 tcgagtaa tttacagtct agactgaccc tttggcag tctagacta tgttaactt 5340 agaagcaatt attgaaccg gtgatcaagt gattcaaaa atcagttca attackca 5400 tattcaagt gtattaaca cagaagtatt tgacccttt gactattgtt attack 5460 aggtaattt tgggaatag agtcagctga agatgttca ggtgatgatg aatttattga 5520 ataagtcgct agaggagaat ggaagttttc taacggcact ttacatattt gtaggatttt 5580 tagcatttta tctctaggt agcacttc aagcattgt acaggctgct gatgcttgtt 5640 gtttatttg gtacacgtgg ttagtattc caggagttaa gggtacagcc tttgtataca 5700 agtatacata tggtagaaaa cttaacaatt cggaattaga agcagttgtt gttaacgagt 5760 ttcctaagaa cggttggaat aaaaaatc cagcaattt tcagatgtc caacgaaca 5820 aattgtactc ttgactttga acagtcagtt gagcttttta aagagtataa tttatta 5880 actgcattct tgttgttctt aaccataata cttcagtag gttatgcaac gcgtagtag 5940 tttattata tacttaaat gatagtgtta tggtgcttt ggccccttaa cattgcagta 6000 ggtgtaattt catgtatata cccaccaac acaggaggtc ttgtcgcagc gataatactt 6060 actgtgtttg cgtgtctttc tttgtaggt tattggatcc agagtattag actctttaag 6120 cggtgtagat cttggtggtc atttaaccca gatctaacg ccgtaggttc atactccta 6180 actaatggtc aacaatgtaa tttgctata gagagtgtgc cgatggtgct ttctcctatt 6240 aaagaatg gtgttcttta ttgtgaggt cagtggcttg ctaaatgtga accagaccac 6300 ttgcctaaag acatattgt atgcacca gatagacgta atatctatcg tatggtgcag 6360 aaatacactg gtgaccaag cggaatag aaaggtttg ctacattgt ctagcaag 6420 cagtcagtag acacctggcga gctagaagt gtagcacag gtggaagtag cctttacaca 6480 taaatgtgtg tgtgtagaga gtatttaaaa ttattctca atagtgccctc tattttaaga 6540 gcgcggaaga gtatttgttt tgaggatatt atataaatc ctctttgttt tgtactctct 6600 ttacaagt tattatta gcacagtttt ttcctttcct tgtttggaa gaaagttgtt 6660 gttaatggtg tagaattcca agtagaaat ggaaaagtcc actacgaagg aaccccatt 6720 ttccaaaaag gttgttgtag gttgtggtcc cattataaga aggattaat ggattaaacc 6780 acctacacta cttacttgta ataagggcgt ttggacttac aagcgcttaa caatacaga 6840 cgatgaaatg gctgactagt ttggaagg cagttattc tgttataaa gccctactat 6900 taactcagtt aagagtatta gataggttaa ttgatca cggaccaag cgagtcttta 6960 cgtgtggtag gcgagtgctt ttatctcaat tagattagt tttaggttg gcatatacgc 7020 ccacccaatc gctggtatga ataatagtaa agataatcct ttcgcggag caatagcaag 7080 aaagcgcga atttactga gagagatt agagtgtgtt tacttctta aaagcagg 7140 acaagcagag ccttgtcccg cgtgtaccctc cctagtattt caggggaaaa cttgtgagga 7200 acacacagat ataataatc tttgtcatg gcgagcggta agacaactgg gagacagac 7260 gccccagcgc cagtcatca actaggaggg ccaaaaccac ctaagttgg ttctctctgga 7320 aatgctagct gtttcaagc actaaaagcc aagaagttaa attcacctcc tcctaagttt 7380 gaaggtagcg gcgttcctga taatgaaat cttaaattta gccagcaaca tgggtactgg 7440 agacgtcaag ccaggtacaa gccaggtaaa ggcggaagaa atcagtccc agatgcttgg 7500 tactttatt acactggaac aggaccagcc gctgacctga attggggtga tagccaagat 7560 ggtatagtgt gggtttctgc aaagggtgct gatactaaat ctagatctaa ccagggtaca 7620 agggatcctg ataagtttga ccaatacccg ctacgattct cagatggagg acctgatggt 7680 aatttccgtt gggacttcat tccaataaat cgtggtagga gtggaagatc aacagcggct 7740 tcatcagcag catctagtag agcaccgtcg cgtgatggct cgcgtggacg tagaagcgga 7800 gctgaagatg atcttatagc tcgtgcagca aagatcattc aggatcagca gaagaagggt 7860 tctcgcatta ctaaagctaa ggccgatgaa atggctcatc gccggtattg taagcgtact 7920 atcccacctg gttataaggt tgatcaagta tttggtcccc gtactaaaagg taaggaggga 7980 aattttggtg atgacaagat gaatgaggag ggtattaagg atgggcgcgt tacagcaatg 8040 ctcaacctag tccctagcag ccatgcttgt ctttttggaa gtagagtgac gcccaaactt 8100 caaccagatg ggctgcactt gagatttgaa tttactactg tggtttctag ggatgatccg 8160 cagtttgata attatgtgaa aatttgtgat cagtgtgtcg atggtgtagg gactcggcca 8220 aaagacgatg aaccgagacc aaagtcacgc ccaaattcaa gacctgctac aagaacaagt 8280 tctccagcgc caagacaaca gcgtcaaaag aaggaagaaga agtcaaagaa gcaggatgat 8340 gaagtagata aggcattgac ctcagatgag gagaggaaca atgcacagct ggaatttgat 8400 gatgaaccga aagtgattaa ctgggggat tcagcacttg gagagaatga gttgtaaagc 8460 tagatttcca acttaacatc atggacgtgc gtatgctgtt tttccctact atagactttt 8520 tagcatatta ttttttgcta tttgtatggt ttattacagg tgaagattgt atgtatttgt 8580 tgtacactcg tatgttctat attatgtttt ctgtagttgt tattagtgtt gttcttgttc 8640 ttactctact gttctctttt ctttatttta gagtatcaat aagaatcaag gaagataggc 8700 atgtagtttg attacctaca tgtctatcgc cagggaaatg tctaatctgt ctacttagta 8760 gcctggaaac gaacggtaga cccttagatt ttaatttagt ttaattttta gtttagttta 8820 agttagttta gagtaggtat aaagaagcca gtgccggggc cacgcggagt acgatcgagg 8880 gtacagcact aggacgccca ctaggggaag agctaaattt tagtttaagt taagtttaat 8940 tggctaagta tagttaaaat ttataggcta gtatagagtt agagcaaaaa aaaaaaaaaa 9000 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 9060 aaaaaaaaaa aaaaaaaaaa aaaaagttta aacttaatta agaattccct tggctcgagt 9120 tcgaaatcgg atgccgggac cgacgagtgc agaggcgtgc aagcgagctt ggcgtaatca 9180 tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga 9240 gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt 9300 gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga 9360 atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc 9420 actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg 9480 gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc 9540 cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc 9600 ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga 9660 ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc 9720 ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat 9780 agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg 9840 cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc 9900 aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga 9960 gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact 10020 agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt 10080 ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag 10140 cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg 10200 tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa 10260 aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata 10320 tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg 10380 atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata 10440 cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg 10500 gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct 10560 gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt 10620 tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc 10680 tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga 10740 tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt 10800 aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc 10860 atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa 10920 tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca 10980 catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca 11040 aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct 11100 tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc 11160 gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa 11220 tattattgaa gcatttatca gggttatattgt ctcatgagcg gatacatatt tgaatgtatt 11280 tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc 11340 taagaaacca ttattatcat gacattaacc tataaaaata ggcgtatcac gaggcccttt 11400 cgtc 11404 <210> twenty two <211> 52 <212> DNA <213> Artificial sequence <220> <223> Primers <400> twenty two cagagcacaa gtttgatctt gtgatatctg atatgtatac agacaatgat tc 52 <210> twenty three <211> 32 <212> DNA <213> Artificial sequence <220> <223> Primers <400> twenty three tatcatagag caaagcacta catagtgcac ac 32 <210> twenty four <211> 38 <212> DNA <213> Artificial sequence <220> <223> Primers <400> twenty four actatgtagt gctttgctct atgataataa tacttacg 38 <210> 25 <211> 40 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 25 cataccaagg ccatttaata taagttttga gaattgagag 40 <210> 26 <211> 31 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 26 aacttatatt aaatggcctt ggtatgtgtg g 31 <210> 27 <211> 48 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 27 cttaactcct ggaattacta accacgtgta ccaaaataaa caacaagc 48 <210> 28 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 28 tcagcatgga cgtgtggtta 20 <210> 29 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 29 aggttggcac ctatatgggg 20 <210> 30 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 30 tgacttggtt tgaagatggc 20 <210> 31 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 31 aagagatgtt ggtaacacct 20 <210> 32 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 32 gacagagcac aagtttgatc 20 <210> 33 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 33 ggagtgaaaa caagatcacc 20 <210> 34 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 34 aatttaacag ttagcgtatc 20 <210> 35 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 35 tttgtatacg agagccatca 20 <210> 36 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 36 ggtcctacta gatgtaaggg 20 <210> 37 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 37 ctctctttga cctacaccat 20 <210> 38 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 38 ttgccttcag tatgtttgtg 20 <210> 39 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 39 agtgaagaaa gtctacctgt 20 <210> 40 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 40 atttcctccg tacttcaaga 20 <210> 41 <211> twenty two <212> DNA <213> Artificial sequence <220> <223> Primers <400> 41 tgaagataat aatggcaaaa gc 22 <210> 42 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> Primers <400> 42 tcttgaaaca ctctcaattc t 21 <210> 43 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 43 ggtcaccagt atatttctgc 20 <210> 44 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> Primers <400> 44 ggtcaacaat gtaattttgc t 21 <210> 45 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 45 cttgtcctgc tttgttaaga 20 <210> 46 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 46 ttataggttg gcttgtacgc 20 <210> 47 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 47 gcccatcctt aataccttcc 20 <210> 48 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 48 ctcgcattac aaaggctaag 20 <210> 49 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> Primers <400> 49 gctctaactc tatactagcc t 21 <210> 50 <211> 32 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 50 gtttgtccaa catctcttac cagtaactta cc 32 <210> 51 <211> 35 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 51 ttactggtaa gagatgttgg acaaaccgct tttac 35 <210> 52 <211> 40 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 52 ggactttgga tcattaaaca gactttttag gtctgtattg 40 <210> 53 <211> 32 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 53 aaagtctgtt taatgatcca aagtcccact ag 32 <210> 54 <211> 32 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 54 acttcaccaa catctcttac cagtaactta cc 32 <210> 55 <211> 31 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 55 ttactggtaa gagatgttgg tgaagtcact g 31 <210> 56 <211> 32 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 56 aatcaaacaa attagcacta catagtgcac ac 32 <210> 57 <211> 41 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 57 actatgtagt gctaatttgt ttgattctga taataattat g 41 <210> 58 <211> 46 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 58 cataccaagg ccacttaata taagttttaa ttattgaaag ttcttc 46 <210> 59 <211> 31 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 59 aacttatatt aagtggcctt ggtatgtgtg g 31 <210> 60 <211> 48 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 60 cagagcacaa gtttgatctt gtgatatctg atatgtatac agacaatg 48 <210> 61 <211> 32 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 61 acttccccaa catctcttac cagtaactta cc 32 <210> 62 <211> 31 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 62 ttactggtaa gagatgttgg ggaagtcact g 31 <210> 63 <211> 47 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 63 cttaactcct ggaattacta accacgtgta ccaaaataaa caacaag 47 <210> 64 <211> 32 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 64 tatcaaacaa agcagcacta catagtgcac ac 32 <210> 65 <211> 36 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 65 actatgtagt gctgctttgt ttgataataa tgaaac 36 <210> 66 <211> 40 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 66 cataccaagg ccatttaata taagtcttga gtattgaaag 40 <210> 67 <211> 31 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 67 gacttatatt aaatggcctt ggtatgtgtg g 31 <210> 68 <211> 32 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 68 tgtcatataa attagcacta catagtgcac ac 32 <210> 69 <211> 38 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 69 actatgtagt gctaatttat atgacaacga atcttttg 38 <210> 70 <211> 40 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 70 cataccaagg ccacttaata taagttttga gtattgaaag 40 <210> 71 <211> 32 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 71 tatcaaacag agcagcacta catagtgcac ac 32 <210> 72 <211> 34 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 72 actatgtagt gctgctctgt ttgataataa tcag 34 <210> 73 <211> 32 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 73 gttgaaccaa catctcttac cagtaactta cc 32 <210> 74 <211> 35 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 74 ttactggtaa gagatgttgg ttcaacctct tttac 35 <210> 75 <211> 32 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 75 tattgtacaa agaagcacta catagtgcac ac 32 <210> 76 <211> 38 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 76 actatgtagt gcttctttgt acaataatga tagctatg 38 <210> 77 <211> 46 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 77 cataccaagg ccatttaata taagttttta aaatagaaag tgtttc 46 <210> 78 <211> 27646 <212> DNA <213> Avian infectious bronchitis virus <400> 78 actaaaaata gatattaata tatatctatt acactagcct tgcgctagat ttccaactta 60 acaaaacgga cttaaatacc tacagctggt cctcataggt gttccattgc agtgcacttt 120 agtgccctgg atggcacctg gccacctgtc aggtttttgt tattaaaatc ttattgttgc 180 tggtatcact gcttgttttg ccgtgtctca ctttatacat ccgttgcttg ggctacctag 240 tatccagcgt cctacgggcg ccgtggctgg ttcgagtgcg aagaacctct ggttcatcta 300 gcggtaggcg ggtgtgtgga agtagcgctt cagacgtact ggttctgttg cgtgaaacgc 360 ggggtcacct ccccccacat acctctaagg gcttttgagc ctagcgttgg gctacgttct 420 cgcacaaggt cggctatacg acgtttgtag ggggtagtgc caaacaaccc ctgaggtgac 480 aggttctggt ggtgtttagt gagcagacat acaatagaca gtgacaacat ggcttcaagc 540 ctaaaacagg gagtatctcc caaaccaagg gatgtcattc ttgtttccaa agacattccc 600 gaacaactct gtgacgcttt atttttctac acgtcacaca accctaagga ttacgctgat 660 gcttttgcat ttaggcaaaa gtttgaccgt aatctgcaga ctgggaagca gttcaaattt 720 gaaactgttt gtggtctctt cctattgaag ggagttgaca aaataacacc tggcgtccca 780 gcaaaagttt taaaagccac ttctaagttg gcagatttag aagacatctt tggtgtctct 840 ccttttgcac ggaagtaccg tgaattgttg aaaacagcat gccagtggtc tcttactgta 900 gaaacactgg atgctcgtgc acaaacgctt gacgaaattt ttgactctac tgaaatactt 960 tggcttcagg tggctgcaaa aattcaagtt tcagctatgg caatgcgcag gcttgttgga 1020 gaagtaactg caaaagtcat ggaagctctt ggctcaaatt tgagtgttct ctttcaaatt 1080 gttaaaac aaatagccag aatctttcaa aaggcactgg ctatttttga aaatgtgagt 1140 gaattaccac agcgtattgc agcacttaag atggcctttg ccaagtgtgc caagtcaatt 1200 actgttgtgg ttgtggaaag aactctagtt gttagagagt tcgcaggaac ttgtcttgca 1260 agcatcaatg gtgctgttgc aaaattcttt gaagaacttc caaatggctt tatgggttct 1320 aaaatcttca caacattggc cttctttaaa gaagcagctg tgaaaattgt ggaaaatata 1380 ccaaatgcac caagaggtac tagaggtttt gaagtcgttg gtaacgccaa gggaacgcaa 1440 gttgttgtgc gtggcatgcg aaatgattta actctgctcg accaaaaagc tgacattcct 1500 gttgagaaag aaggttggtc tgcaattctt gaaggacatc tgtgttatgt ctttaagagt 1560 ggtgatcgtt tttatgcggc acctctttca gggaattttg cattgcatga tgtgcattgt 1620 tgtgagcgtg ttgtctgtct gtctgatggt gtaacaccag agataaatga tggactcatt 1680 ctagcagcaa tctattcatc ttttagtgtc tcagaactcg tggcagcact taaaaagggt 1740 gaccattca agttcttggg tcataaattt gtgtatgcga agtctcttc actcttgcaa aagcagccac tattgcagat gtactgaagc tgtttcaatc agctcgtgtg caaacagaag atgtgtggtc tgcatttact gaaaagtctt ttaatttctg gaaactcgca tatggaaag tgcgtaatct tgaagaagtt gtgaagactc atttttgtaa agctcaaatg tcaattatca ttctagcagc agtgcttggc gaaggcattt ggcatcttgt ttcacaggtc atctaaag tagtggtct ttttactaga gtcgttgact tttgtgaaaa acactggaag ggtttctgtg cacaacttaa aaaggctaag ctcgttgtca cagaaactct ttgtgttctt aagggagtgg cacagcattg ttttcaacta ttgctggatg caatacattc tttgtatatg agttttaaga agtgtgcact tggtagaatt catggagact tactcttctg gaaagggggt gtacacaaaa ttgttcaaga tggcgatgaa gtttggtttg acgccattga tagtattgat gttgaagatc tgggtgttgt ccaagaaaaa cccatagatt ttgaggtttg tgaagacgta acacttccag aaaatcaacc tggtcatatg gttcaaatcg aggatgacgg aaagaactat atgttcttcc gcttcaaaag ggatgagaac atctactata caccaatgtc tcaacttggt 2520 gcaattaatg tagtttgcaa agcaggcggt aaaaccgtta cctttggaga caccattgtg 2580 aaagaaatac cgccacctga tgttgtgcct attaaggtta gcatagagtg ttgtggtgaa 2640 ccatggaata caatcttcaa gaaagcttat aaagagccca ttgaagttga aactgacctc 2700 acagtagaac aattgctctc tgtgatctat gagaaaatgt gcgacgacct caaattgttt 2760 ccagaggcac cagagccacc gccatttgag aatgtcgcac ttgttgataa aaacggtaaa 2820 gacttggatt gcataaaatc atgccatctt atctaccgtg attatgagag tgatgatgac 2880 atcgaggaag aagatgctga ggagtgtgat actgatttag aatgtgaaga agaggatgag 2940 gatactaaag tgttggctct tatacaagac cctgcaagta ataataccc tcttcctct 3000 gatgatgatt atagcgtctt taatggatgt attgtacata aggacgctct tgacgtcgta 3060 aatctaccat ctggtgaaga aacctttgtt gtcaacaact gctttgaggg agctgtaaaa 3120 ccactgcctc agaaagttgt tgatgttcta ggtgactggg gtgaggctgt tgatgcgcaa 3180 gagcaaattg cacaaactac ttcagaggaa acccctatca gtagtttgga ggcaactatt 3240 gagcaagttg ttgttgagga acagaaaata atttctgttg ttgaagaaga acagcaggtg 3300 gcggtctaca cacctgcaga cctacaagtt gttgaagaaa caccagatga gtttattctt 3360 attgctgatg tttccacaga agaaattgtg cctcatgaag aaaaggagtc acagattgaa 3420 caggagccta ttcaagttgt taaatcacaa cgtgaaaaga aggctaaaaa gttcaaggtt 3480 aaatctacta catgtgagaa acccaaattt ttggagtaca caacatgtgt gggtgaccta 3540 acggtagtga ttgccaaagc attggatgag tttaaagagt tctgcattgt aaatgctgct 3600 aatgagcata tgtctcacgg tggcggcgtt gcaaaggcaa ttgcagactt ttgtggaccg 3660 gactttgttg aatattgcgc ggactatgtt aagaaacatg gtccacagca aaaacttgtc 3720 acaccttcat ttgttaaagg cattcaatgt gtgaataatg ttgtaggacc tcgccatgga 3780 gacagcaact tgcgtgagaa gcttgttgct gcttacaaga gtgttcttgt aggtggagtg 3840 gttaactatg ttgtgccagt tctctcatca gggatttttg gtgtagattt taaaatgtca 3900 atagatgcta tgcgcgaagc ttttaaaggt tgtgccatac gcgttctttt atttctctg 3960 agtcaagaac acatcgatta tttcgatgca acttgtaagc agaagacaat ttatcttacg 4020 gaggatggtg ttaaataccg ctctgttgtt ttaaaacctg gtgattcttt gggtcaattt 4080 ggacaggtt ttgcaagaaa taaggtagtc tttcggctg atgatgttga ggataaagaa 4140 atcctcttta tacccacaac tgacaagact attcttgaat attatggtttt agatgcgcaa 4200 aagtatgtaa tatatttgca aacgcttgcg cagaaatggg atgttcaata tagagacaat 4260 tttgttatat tagagtggcg tgacggaaat tgctggatta gttcagcaat agtgctcctt 4320 caagctgcta agattaggtt taaaggtttt cttgcagaag catgggcaca acttttgggt 4380 ggagacccaa ctgatttgtgt agcctggtgc tatgcaagtt gcaatgctaa tgttggtgag 4440 ttttcagatg ctaattggct tcttgctaat ttggcagaat actttgatgc tgattacacg 4500 aatgcattcc ttaagaggcg tgtgtcatgt aactgtgggg ttaagaattg tgaagttaga 4560 ggccttgaag cttgtattca accagtaaag gcacccaatc ttcttcattt caagactcag 4620 tacaaaatt gtacagtgtg tgatgcaaat agtgtggatg aggtggtaga ggcctcacta 4680 ccatatctgt tgctccttgc tactgatggt cctactacag tggattgtga tgaaaatgct 4740 gtagggaatg ttgttttcat tggctctact aatagtggcc attgttacac gcaagccatt 4800 ggtaaggctt ttgataatct tgctaaggat agaaaatttt caaagaattc gccataacatt 4860 acagcaatgt atacgcgctt ctctcttaag agtgaaagct ctctgtctgt tgttaaacag 4920 agtaagagta aaactaaagt agtaaaagaa gatgttgcca accttgttac tagttctaaa 4980 gccagttttg atgactcttac tgactttgaa cattggtatg atagtaacat ctatgaaagt 5040 cttaaagttc aggaaatacc tgtgaatttg gatgagtatg tgtcatttac aacgaaagaa 5100 gactataagt tgccactgac acttaaagtt agaggtatca aatcagttgt tgactttatt 5160 tcaagagacg gtttctctta tagttaaca cctgacattg aagaaaattc aaaagcgcca 5220 gtctactacc cagtcttaga ctctattagt cttaaggcaa tatgggtaga cggcagtgct 5280 aattttgttg ttggtcatcc aaactactat agtaagtctc ttcgcattcc tacttttgg 5340 gaaaatgcag agagctttgt taagataggt gacaaagttg atggtgtaac tatgggcctt 5400 tggcgtgcag aacagcttaa caaacctaat cttgaaagaa tttcaacat tgctaagaaa 5460 gctattgttg gatccagtgt tgttactaca caatgtagta aattaattag taaagcagct 5520 acattcattg ctgataaagt aggtgggggt gtagttcgta atattacaga tagaattaag 5580 ggtctttgtg gatttacacg tgggcatttt gaaagaaaat tgtctccaca attcataaaa 5640 acacttatat tcttcttctt ttactttgta aaggctagtg ctaagagtgt tgccactagt 5700 tataagcgtg tgttatgtaa ggtggtttt accacgctat ttatattatg gtttatgtac 5760 acaagcaaac cagtaacttt tactggaaca cgtgtgctag acttcttatt tgagggttct 5820 tttgtggtc cctataatga ctatggtaaa gactcatttg acgtactacg ctattgtgga 5880 gatgattta cttgtcgtgt atgtttacat gataaagatt cacttcattt gtataagcat 5940 gcttatagcg tagaacaggt ttataaagat gcagcttctg gcattagttt taattggaat 6000 tggctttatt tggtctttct aattattt gttaaaccag tagcaggttt cgttattatt 6060 tgctattgtg ttagtactt ggtattgagt tcaactgtgt tgcaaactgg tgtaggtttt atggactggt ttattcaaac agttttct cactttaatt ttattgggc aggttctat ttctggctct tttataaatt gtacatacag gttcatcata tactgtattg tagtata acatgtgaag tgtgtaagag agttgcacgc agtaacaggc atgaggttag tgttgttgtt ggtggacgca agcaaattgt gcatgtgtac actaactctg gttacaactt ttgtaagaga cataattggt attgtaggaa ttgtgatgta tatggtcacc aaaacacatt tatgtctcct gaagttgctg gcgagctttc tgaaaagctt aaacgccatg ttaaacctac agcacatgct taccacgttg tggatgaggc ttgcgtagtt gatgattttg ttaacttaaa atacaaagct gcaactcctg gtaaggatgg tgcacctcct gcagttaaat gtttcagtgt tacagatttc ttgaagaaag ctgtttttct tag ctgaaatgtg aacaaatatc tag tttatagtgt gtaatacgca gagtgcgcat gctttagagg aagcaaagaa tgcagccatc tattatgcgc aatacctgtg taaacctata cttatactcg accaggcact ctaccagaat ttaatagtgg aacctgtatc gaagagcgtt gtcaacaaag tgtgtgacat tttgtctagg 6840 ataatttctg tagatactgc atctttggat tataaagcag gtacaattcg tgatgccttg 6900 ctgtctgtta ctaaagatga agaagctgta gatatggcta tcttctgtca taatcatgaa 6960 gttgaatata caggtgatgg ttttactaat gttataccgt catatggtat agacactgat 7020 aaattaacac ctcgtgatag agggttttg ataaatgcag atgcttctgt tgctaactta 7080 agagttaaaa atgctccgcc ggtagtatgg aagttctctg atcttattaa gttgtctgac 7140 agttgtctta aatatttaat ctcagcaact gtcaagtcag ggtctcgttt ctttataaca 7200 agatctggtg ctaaacaaat ttttctttgt agtactcaga aattgttggt agagaaaaag 7260 gctggtggtg tcattagtgg tacctttaat tggtttaaga gttgttgtaa atggctcttg 7320 atcttctatg tgctttttac attgtgttgt ttgggttgtt atcatatgga gacgaataaa 7380 agttttgttc atcctatgta tgatgttaac tctacaatgc atgttgaagg ctttaaggtt 7440 atagataaag gtgttattag agacattgta ccagaggatg cttgtttctc tataagtttt 7500 gctaactttg atgcattttg gggtaaacca tatgtgaata gtagagactg tccaattgtt 7560 acagcagtca tagatggcgc tggaacaata gcagctggtg ttcctggttt tgtagactgg 7620 gttcttgatg gtgttatgtt tgtacacatg acacaaacag aaagaaaacc ctggtacatt 7680 cccacgtggt ttaacagaga aattgttggt tacactcagg attcaattat tactgaaggt 7740 agtttttata catctatagc tttgttttca gctaggtgtt tatatttaac agccagcaat 7800 acaccacaat tgtattgttt taatggtcat aatgatgctc ctggagcctt accatttagc 7860 agtatcactc cacacagggt ctacttccaa ccaaatggtg ttaggcttat aattcctcaa 7920 cagataatgc acacacccta cgtagtaaag tttttatcag acagctattg tagaggtagt 7980 gtatgtgagt atactaaacc gggttattgt gtctcactaa attcccaatg ggttttattt 8040 aatgacgaat acacaagtaa accaggagta ttctgtggtt ctactgttag agaacttatg 8100 tttaatatgg ttagtacatt ttttactggt gtcaacccta atatttatat gcagctggcg 8160 actatgttct tatactagt tgttgttgtg ttaatttttg caatggttat aaagtttcaa 8220 ggtgttttta aagcttatgc aaccattgtg tttacaataa tgctagtttg ggttgttaat 8280 gcatttattt tgtgtgtaca tagttataat agtgttgtgg ctgttatact actagtaatc 8340 tattgttatg catcattggt tacaagtcgt aatactgcta tataatgca ttgttggctt 8400 gtgttacct ttggtttaat tgtacccata tggttggcgt gttgctacct ggcatttgtt 8460 ttatatatgt acacaccatt gttttctgg tgttacggta ctactaaaaa cactcgtaaa 8520 ttgtatgatg gcaacgagtt tgttggtact tatgatcttg ctgcgaagag cacctttgtt 8580 attcgcggtc ctgaatttgt taagcttacg aacgagatag gtgataagtt tgaacactat 8640 ctctcagcgt atgctagact taatactac tcaggcactg gcagtgaaca agattatctg 8700 caagcttgtc gtgcatggtt agcttatgct ttggaccaat atagaatag tggtgtggaa 8760 attgtgtata ctccaccacg ttactctatt ggtgttagta gattacaggc cggttttaag 8820 aaactagttt ctcctagtag tgttgttgaa aagtgcattg ttagtgtctc ttatagaggt 8880 aataatctta atggactgtg gctaggtggt actatctact gtccgcgaca tgttctaggc 8940 aagttctcag gtgaccaatg gatgatgta cttaaccttg ctaataatca tgagtttgaa 9000 gttgtaactc aaataatgt tactttgaat gttgtcagta ggaggctaaa agttgcagtt 9060 ttgattttac aaactgctgt tgctaatgct gaaactccaa agtataagtt tgttaaagcc 9120 aattgtggag atagttttac gattgcttgt tcctatggtg gtacagttgt tggacttac 9180 cctgttacta tgcgctctaa tgtactatt agggcgtctt tcttagcagg agcgtgtggt 9240 tctccaggtt ttatataga aaagggtgta gttaactttt attatatgca ccatcttgag 9300 ttgcctaatg cattacacac aggactgac ctaatgggag agttctatgg tggttatgtg 9360 gacgaagagg ttgcacaag ggtgccacca gataatttag ttactacaa tattgtagca 9420 tggctttatg ccgcaattat tagtgttaag gagagtagtt tctcactgcc taaatggttg 9480 gatagtacta ctgtcagtgt tgaagactgc ataagtggg ctggtgataa tggttttaca 9540 ccattttcta ctagtactgc tattactaaa ttaagtgcta taacaggagt agatgtttgt 9600 aaactccttc gcacttat gggtaaaagt agtcaatggg gtagtgatcc cattttagga 9660 caataatt ttgaagatga attgacacca gagtccgttt ttaatcagat aggtggtgtt 9720 agattacagt cttctttgtgt aagaagagcc acatcctggt tttggagtag atgtgtgtta 9780 gcttgcttct tatttgtgtt gtgtgctatt gtcttgtta cggcagtgcc acttaaatat 9840 tatgtacatg cagccgttat tttgttaaca gctgtgctct tcatttcttt tactgttaaa 9900 catgtatgg catatatgga tacttttcta ctgccaacat tgcttacagt cattattgga 9960 gttgtgctg aagtaccttt catctacaat actctaatta gtaggatagt tgtctttgtt 10020 agtcaatggt atgatcctgt agtctttgat actatggtac catggatgtt cttgccacta 10080 gtgttgtaca cagcatttaa gtgtgtgcag ggttgctata gtgtgaattc tttcaatact 10140 tctttgctag tactgtacca gttcttgaag ttaggctttg ttatttatgc ctcttctagc 10200 acgctggcag catacacaga aggtaattgg gatttatttt ttgaattagt tcacactact 10260 gtgttggcta atgttagtag taattcctta ataggtttgt ttgtgttcaa gttagctaag 10320 tggatgttgt attattgtaa tgctacatac tttaataatt atgtgctaat ggctgtcatt 10380 attaatggct ttggttggct cttcacttgt tactttggag tttattggtg gattaataag 10440 gttttggtt taaccttagg taaatatgaa ttcaaagtttt cagtagacca atataggtat 10500 atgtgtcttc ataagataaa ttcgcctaaa actgtgtggg aagttttttc gacaaatata 10560 cttatacaag gaattggtgg tgatcgtgtg ttgcctatag ctacagtgca atctaaattg 10620 agtgatgtaa agtgtacaac tgttgtttta atgcagcttt tgactaagct taatgttgaa 10680 gcaaattcaa aaatgcatgc ttatcttgtt gagttacaca ataaaatcct cgcatctgat 10740 gatgttggag agtgcatgga taatttattg ggtatgctta taacactatt ttgtatagat 10800 tctactattg atttgggtga gtattgtgat gatatactta agaggtcaac tgtattacaa 10860 tcggttactc aagagttttc gcacataccc tcgtatgctg aatatgaaag agctaagagt 10920 attatgaaa aggttttagc cgattctaaa aatggtggtg taacacagca agagcttgct 10980 gcatatcgta aagctgccaa tattgcaaag tcagtttttg atagagactt ggctgttcaa 11040 aagaagttag atagcatggc agaacgtgct atgacaacaa tgtataaaga ggcgcgtgta 11100 actgatagaa gagcaaaatt agttcatca ttacatgcac tactttttttc aatgcttaag 11160 aaatagatt ctgagaagct taatgctta tttgaccagg cgaatagtgg tgttgtaccc 11220 ctagcaactg ttccaattgt ctgtagtaat aagcttaccc tcgtatacc agacccagag 11280 acgtgggtca agtgtgtgga gggtgtgcat gttacatatt caacagttgt ttggaatata 11340 gactgtgtta ctgatgccga tggcacagag ttacacccca cttctacagg tagtggattg 11400 acttactgta taagtggtga tatatagca tggcctttaa aggttaactt gactaggaat 11460 gggcataata aggttgatgt tgccttgcaa aataatgagc ttatgcctca cggtgtaaag 11520 acaaaggctt gcgtagcagg tgtagatcaa gcacattgta gcgttgagtc taaatgttat 11580 tatacaagta ttagtggcag ttcagttgta gctgctatta cctcttcaaa tccaaatctg 11640 aaagtagcct cttttttgaa tgaggcaggt aatcagattt atgtagactt agacccacca 11700 tgtaaatttg gtatgaaagt gggtgataag gttgaagttg tttacctgta ttttataaaaa 11760 aatacgaggt ctattgtaag aggtatggta cttggtgcta tatctaatgt tgttgtgtta 11820 caatctaaag gtcatgagac agaggaagtg gatgctgtag gcattctctc actttgttct 11880 tttgcagtag atcctgcgga tacatattgt aaatatgtgg cagcaggtaa tcaaccttta 11940 ggtaactgtg ttaaaatgtt gacagtacat aatggtagtg gttttgcaat aacatcaaag 12000 ccaagtccaa ctccggatca ggattcttat ggaggagctt ctgtgtgtct ttattgtaga 12060 gcacatatag cacaccctgg cggagcagga aatttagatg gacgctgtca atttaaaggt 12120 tcttttgtgc aaatacctac tacggagaaa gatcctgttg gattctgtct acgtaacaag 12180 gtttgcactg tttgtcagtg ttggattggt tatggatgtc agtgtgattc acttagacaa 12240 cctaaacctt ctgttcagtc agttgctgtt gcatctggtt ttgataagaa ttatttaaac 12300 gggtacgggg tagcagtgag gctcggctga taccccttgc taatggatgt gaccccgatg 12360 ttgtaaagcg agcctttgat gtttgtaata aggaatcagc cggtatgttt caaaatttga 12420 agcgtaactg tgcacgattc caagaagtac gtgatactga agatggaaat cttgagtatt 12480 gtgattctta ttttgtggtt aaacaaacca ctcctagtaa ttatgaacat gagaaagctt 12540 gttatgaaga cttaaagtca gaagtaacag ctgatcatga tttctttgtg ttcaataaga 12600 acatttataa tattagtagg cagaggctta ctaagtatac tatgatggat ttttgctatg 12660 ctttgcggca ctttgaccca aaggattgcg aagttcttaa agaaatactt gtcacttatg 12720 gttgtataga agattatcac cctaagtggt ttgaagagaa taaggattgg tacgacccaa 12780 tagaaaaccc taaatattat gccatgttgg ctaaaatggg acctattgta cgacgtgctt 12840 tattgaatgc tattgagttc ggaaacctca tggttgaaaa aggttatgtt ggtgttatta 12900 cacttgataa ccaagatctt aatggcaaat tttatgattt tggtgatttt cagaagacag 12960 cgcctggtgc tggtgttcct gtttttgata cgtattattc ttacatgatg cccatcatag 13020 ccatgactga tgcgttggca cctgagaggt attttgaata tgatgtgcat aagggttata 13080 aatcttatga tctcctcaag tatgattata ctgaggagaa acaagagttg tttcagaagt 13140 actttaagta ttgggatcaa gagtatcacc ctaactgtcg cgactgtagt gatgacaggt 13200 gtttgataca ttgtgcaaac ttcaacatct tgttttctac acttgtaccg cagacttctt 13260 tcggtaattt gtgtagaaag gtttttgttg atggtgtacc atttatagct acttgtggct 13320 atcattctaa ggaacttggt gttattatga atcaagaataa caccatgtca ttttcaaaaa 13380 tgggtttgag tcaactcatg cagtttgttg gagatcctgc cttgttagtg gggacatcca 13440 ataaattagt ggatcttaga acgtcttgtt ttagtgtttg tgctttagcg tctggtatta 13500 ctcatcaaac ggtaaaacca ggtcacttta acaaggattt ctacgatttt gcagagaagg 13560 ctggtatgtt taggaaggt tcttctatac cacttaaaca tttcttctac ccacagactg 13620 gtaatgctgc tataaacgat tatgattatt atcgtttaaa caggcctacc atgtttgata 13680 13740 gctgtatacc agcaagccaa gttgtagtta acaatttaga taagagtgca ggttatccgt 13800 tcaataagtt tggaaggcc cgtctctatt atgaaatgag tctagaggag caggaccaac 13860 tctttgagag tacaaagaag aacgtcctgc ctactataac tcagatgaat ttaaaatatg 13920 ccatatccgc gaaaaataga gcgcgtacag tggcaggtgt gtctatcctt tctactatga 13980 ctaataggca gtttcatcag aagattctta agtctatagt caacactaga aacgctcctg 14040 tagttattgg aacaaccaag ttttatggcg gttgggataa catgttgaga aaccttattc 14100 agggtgttga agacccgatt cttatgggtt gggattatcc aaagtgtgat agagcaatgc 14160 ctaatttgtt gcgtatagca gcatctttag tactcgctcg taaacacact aattgttgta 14220 cttggtctga acgcgtttat aggttgtata atgaatgcgc tcaggtttta tctgaaactg 14280 tcttagctac aggtggtata tatgtgaaac ctggtggtac tagcagtgga gatgctacta 14340 ctgcttatgc aaacagtgtt ttcaacataa tacaagccac atctgctaat gttgcgcgtc 14400 ttttgagtgt tataacgcgt gatattgtat atgatgacat taagagcttg cagtatgaat 14460 tgtaccagca ggtttatagg cgagtcaatt ttgacccagc atttgttgaa aagttttatt 14520 cttattgtg taagaatttc tcattgatga tcttgtctga cgacggtgtt gtttgtta 14580 acaacacatt agccaaacaa ggtcttgtag cagatatttc tggttttaga gaagttctct 14640 actatcagaa caatgttttt atggctgatt ctaaatgttg ggttgaacca gatttagaaa 14700 aaggcccaca tgaattttgt tcacagcaca caatgttagt ggaggttgat ggtgagccta 14760 gatacttgcc atatccagac ccatcacgta ttttgtgtgc atgtgttttt gtagatgatt 14820 tggataagac agaatctgtg gctgttatgg agcgttatat cgctcttgcc atagatgcgt 14880 acccactagt acatcatgaa aatgaggagt acaagaaggt attctttgtg cttctttcat 14940 acatcagaaa actctatcaa gagctttctc agaatatgct tatggactac tctttttgtaa 15000 tggatataga caagggtagt aaatttggg agcaggagtt ctatgaaaat atgtatagag 15060 cccctacaac attacagtct tgtggcgttt gtgtagtgtg tatagtcaa actatattgc 15120 gctgtggtaa ttgtattcgc aaaccatttt tgtgttgtaa gtgttgctat gaccatgtca 15180 tgcacacaga ccacaaaaat gttttgcta taaatcctta catttgctca cagccaggtt 15240 gtggtgaagc agatgttact aaattgtacc tcggaggtat gtcatacttc tgcggtaatc 15300 ataaaccaaa gttatcaata ccgttagtat ctaatggtac agtgtttgga atttacaggg 15360 ctaattgtgc aggtagcgaa aatgttgatg attttaatca actagctact actaattggt 15420 ctactgtgga accttatatt ttggcaaatc gttgtgtaga ttcgttgaga cgctttgctg 15480 cagagacagt aaaagctaca gaagaattac ataagcaaca atttgctagt gcagaagtga 15540 gagaagtact ctcagatcgt gaattgattc tgtcttggga gccaggtaaa accaggcctc 15600 cattgaatag aaattatgtt ttcactggct ttcactttac tagaactagt aaagttcagc 15660 tcggtgattt tacatttgaa aaaggtgaag gtaaggacgt tgtctattat cgagcgacgt 15720 ctactgctaa attgtctgtt ggagacattt ttgttttaac ctcacacaat gttgtttctc 15780 ttatagcgcc aacgttgtgt cctcagcaaa ccttttctag gtttgtgaat ttaagaccta 15840 atgtgatggt acctgcgtgt tttgtaaaca acattccatt gtaccattta gtaggcaagc 15900 agaagcgtac tacagtacaa ggccctcctg gcagtggtaa atcccatttt gctataggat 15960 tggcggctta ctttagtaac gcccgtgtcg ttttactgc atgctctcat gcagctgttg 16020 atgctttatg tgaagaagct tttaagtttc ttaaagtaga tgattgcact cgtatagtac 16080 ctcaaaggac tactatcgat tgcttctcta agtttaaagc taatgacaca ggcaaaaagt 16140 acatttttag tactattaat gccttgccag aagttagttg tgacattctt ttggttgacg 16200 aggttagtat gttgaccaat tacgaattgt cttttattaa tggtaagata aactatcaat 16260 atgttgtgta tgtaggtgat cctgctcaat taccggcgcc tcgtacgttg cttaacggtt 16320 cactctctcc aaaggattat aatgttgtca caaaccttat ggtttgtgtt aaacctgaca 16380 ttttccttgc aaagtgttac cgttgtccta aagaaattgt agatactgtt tctactcttg 16440 tatatgatgg aaagtttatt gcaaataacc cggaatcacg tcagtgtttc acggttatag 16500 ttaataatgg taattctgat gtaggacatg aaagtggctc agcctacaac acaactcaat 16560 tagaatttgt gaaagatttt gtctgtcgca ataaggaatg gcgggaagca acattcattt 16620 caccttataa tgctatgaac cagagagcct accgtatgct tggacttaat gttcagacag 16680 tagactcgtc tcaaggttcg gagtatgatt atgttatctt ttgtgttact gcagattcgc 16740 agcatgcact gaatattaac agattcaatg tagcgcttac aagagccaag cgtggtatac 16800 tagttgtcat gcgtcagcgt gatgaactat attcagctct taagtttata gagcttgata 16860 gtgtagcaag tctgcaaggt acaggcttgt ttaaaatttg caacaaagag tttagtggtg 16920 ttcatccagc ttatgcagtc acaactaagg ctcttgctgc aacttataaa gtaaatgatg 16980 aacttgctgc acttgttaac gtggaagctg gttcagaaat aacatataaa catcttattt 17040 ctttgttagg gtttaagatg agtgttaatg ttgaaggctg ccacaacatg tttataacac 17100 gtgatgaggc tatccgcaac gtaagaggtt gggtaggttt tgatgtagaa gcaacacatg 17160 cttgcggtac taacattggt actaacctgc ctttccaagt aggtttctct actggtgcag 17220 gctttgtagt tacgcctgag ggacttgtag atacttcaat aggcaataat tttgagcctg 17280 tgaattctaa agcacctcca ggtgaacaat ttaatcactt gagagcgtta ttcaaaagtg 17340 ctaaaccttg gcatgttgta aggccaagga ttgtgcaaat gttagcggat aacctatgca 17400 acgtttcaga ttgtgtagtg tttgtcacgt ggtgtcatgg cctagaacta accactttgc 17460 gctattttgt taaaataggc aaggaccaag tttgttcttg cggttctaga gcaacaactt 17520 ttaattctta tactcaggct tatgcttgtt ggaagcattg cttgggtttt gattttgttt 17580 ataatccact cttagtggat attcagcagt ggggttattc tggtaaccta caatttaacc 17640 atgatttgca ttgtaatgtg catggacacg cacatgtagc ttctgcggat gctattatga 17700 cgcgttgtct tgcaattaat aatgcatttt gtcaagatgt caactgggat ttaacttacc 17760 ctcatatagc aaatgaggat gaagtcaatt ctagctgtag atatttacaa cgcatgtatc 17820 ttaatgcatg tgttgatgct cttaaagtta acgttgtcta tgatataggc aaccctaaag 17880 gtataaaatg tgttagacgt ggagacttaa attttagatt ctatgataag aatccaatag 17940 tacccaatgt caagcagttt gagtatgact ataatcagca caaagataag tttgctgatg 18000 gtctttgtat gttttggaat tgtaatgtgg attgttatcc cgacaattcc ttagtttgta 18060 ggtacgacac acgaaatttg agtgtgttta acctacctgg ttgtaatggt ggtagcttgt 18120 atgtcaacaa gcatgcattc cacacaccta aatttgatcg cactagcttt cgtaatttga 18180 aagctatgcc attctttttc tatgactcat cgccttgcga gaccattcaa gtggatggag 18240 ttgcgcaaga ccttgtgtca ttagctacga aagattgtat cacaaaatgc aacataggcg 18300 gtgctgtttg taaaaagcac gcacaaatgt atgcagattt tgtgacttct tataatgcag 18360 ctgttactgc tggttttact ttttgggtta ctaataattt taacccatat aatttgtgga 18420 aaagtttttc agctctccag tctatcgaca atattgctta taatatgtat aagggtggtc 18480 attatgatgc tattgcagga gaaatgccca ctatcgtaac tggagataaa gtttttgtta 18540 tagatcaagg cgtagaaaaa gcagtttttt ttaatcaaac aattctgcct acatctgtag 18600 cgtttgagct gtatgcgaag agaaatattc gcacactgcc aaacaaccgt attttgaaag 18660 gtttgggtgt agatgtgact aatggatttg taatttggga ttacacgaac caaacaccac 18720 tataccgtaa tactgttaag gtatgtgcat atacagacat agaaccaaat ggcctaatag 18780 tgctgtatga tgatagatat ggtgattacc agtcttttct ggctctctgat aatgctgttt 18840 tagtttctac acaggtgttac aagcggtatt cgtatgtaga aataccgtca aacctgcttg 18900 ttcagaacgg tattccgtta aaagatggag cgaacctgta tgtttataag cgtgttaatg 18960 gtgcgtttgt tacgctacct aacacatta acacacaggg tcgcagttat gaaacttttg 19020 aacctcgtag tgatgttgag cgtgattttc tcgacatgtc tgaggagagt tttgtagaaa 19080 agtatggtaa agaattaggt ctacagcaca tactgtatgg tgaagttgat aagccccaat 19140 taggtggttt acacactgtt ataggtatgt gcagactttt acgtgcgaat aagttgaacg 19200 caaagtctgt tactaattct gattctgatg tcatgcaaaa ttattttgta ttggcagaca 19260 atggttccta caagcaagtg tgtactgttg tggatttgct gcttgatgat ttcttagaac 19320 ttcttaggaa catactgaaa gagtatggta ctaataagtc taaagttgta acagtgtcaa 19380 ttgattacca tagcataaat tttatgactt ggtttgaaga tggcattatt aaaacatgtt 19440 atccacagct tcaatcagca tggacgtgtg gttataatat gcctgaactt tacaaagttc 19500 agaattgtgt tatggaacct tgcaacattc ctaattatgg tgttggaata gcgttgccaa 19560 gtggtattat gatgaatgtg gcaaagtata cacaactctg tcaatacctt tcgaaaacaa 19620 caatgtgtgt accgcatagt atgcgagtaa tgcattttgg agctggaagt gacaaaggag 19680 tggctccagg tagtactgtt cttaaacaat ggctcccaga agggacactc cttgtcgata 19740 atgatattgt agactatgtg tctgatgcac atgtttctgt gctttcagat tgcaataaat 19800 ataagacaga gcacaagttt gatcttgtga tatctgatat gtatacagac aatgattcaa 19860 aaagaaagca tgaaggcgtg atagccaata atggcaatga tgacgttttc atatatctct 19920 caagttttct acgcaataat ttggctctgg gaggcagttt tgctgtaaaa ttaacagaga 19980 caagttggca tgagagttta tatgacattg cacaggattg tgcatggtgg acaatgttct 20040 gtacagcagt gatgcatct tcttcagaag cattcctgat tggtgttaat tacttgggtg 20100 caagtgcaaa ggttaaagtt agtggaaaa cactgcacgc aaattatata ttttggagga 20160 attgtaatta tttacaacc tctgcttata gtatatttga tgttgctaag tttgatttga 20220 gattgaaagc aacgccagtt gttaatttga aactgaaca aaagacagac ttagtcttta 20280 atttaattaa gtgtggtag ttactgtaa gagatgttgg taacactct tttactgtg 20340 actctttgt gtgcactatg tagtgctgtt ttgtatgaca gtagttctta cgtgtactac 20400 taccaaagtg ccttcagacc acctgatggt tggcatttac atggggtgc gtatgcggtt 20460 gttaatattt ctagtgaatc taataatgca ggctcttcat ctgggtgtac tgttggtatt 20520 attcatggtg gtcgtgttgt taatgcttct tctatagcta tgacggcacc gtcatcaggt 20580 atggcttggt ctagcagtca gttttgtact gcatactgta acttttcaga tactacagtg 20640 ttgttacac attgttataa acatgttggg tgtcctataa ctggcatgct tcacagcat 20700 tctatacgtg tttctgctat gaaaaatggc cagccttttt ataatttaac agttagtgta 20760 gctaagtacc ctacttttaa atcattcag tgtgttaata atttacac cgtatatta 20820 aatggtgatc ttgtttacac ctctaatgag accacagatg ttacatctgc aggtgtttat 20880 tttaaagctg gtggacctat aacttataaa gttatgagag aagttagagc cctggcttat 20940 tttgttaatg gtactgcaca agatgttatt ttgtgtgatg ggccacctag aggcttgtta 21000 gcatgccagt atatactgg caattttca gatggctttt atccttttac taatagtagt 21060 ttagttaagc agaagtttat tgtctatcgt gaaatagtg ttaatactac ttttacgtta 21120 cgcaatttca cttttcataa tgagactggc gccaacccta atcctagtgg tgttcagaat 21180 attcaaactt accaacaca aacagctcag agtggttatt ataattttta ttttccttt 21240 ctgagtagtt ttgtttataa ggagtctaat tttagtatg gatcttatca cccaagttgt 21300 aattttagac tagaactat taataatggt ttgtggttta attcactttc agttttcatt 21360 gcttacggtc ctctcagg tggttgcaag caatctgtct ttagtgtag agcaacctgt 21420 tgttatgctt actcatatgg aggtcctttg ctgtgtaaag gtgtttattc aggtgagtta 21480 ggtcataatt ttgaatgtgg actgttagtt tatgttacta agagcggtgg ctctcgtata caaacagcca ctgaaccgcc agttataact caacacaatt fathers tactttaaat acttgtgttg attack attack actggccaag gttttattac attackac gactcagctg ttagttataa ttatctagca gacgcaggtt tggctatttt agatacatct ggttccatag acatctttgt tgtacaaggt gaatggtc ttacttatta taaggttaac ccttgcgaag atgtcaacca gcagtttgta gtttctggtg gtaaattagt aggtattctt acttcacgta atgagactgg ttctcagctt cttgagaacc agttttacat taaaatcact aatggaacac gtcgttttag acgttctatt actgaaaatg ttgaaaattg cccttatgtt agttatggta agttttgtat aaaacctgat ggttcaattg ccacaatagt accaaaacaa ttggaacagt ttgtggcacc tttacttaat gttactgaaa atgtgctcat acctaacagt tttaatttaa ctgttacaga tgagtacata caaacgcgta tggataaggt ccaaattaat tgtctgcagt atgtttgtgg caattctctg gattgtagag atttgtttcg acaatatggg 22200 catgtttgtg acaacatatt gtctgtagta aatagtattg gtcaaaaaga agatatggaa 22260 cttttgaatt tctattcttc tactaaaccg gttggtttta atacaccatt tcttagtaat 22320 gttagcactg gtgagtttaa tatttctctt ctgttaacaa ctcctagtag tcctagaagg 22380 cgttctttta ttgaagacct tctatttaca agcgttgaat ctgttggatt accaacagat 22440 gacgcataca aaaattgcac tgcaggacct ttaggttttc ttaaggacct tgcgtgtgct 22500 cgtgaatata atggtttgct tgtgttgcct cccattataa cagcagaaat gcaaactttg 22560 tatactagtt ctctagtagc ttctatggct tttggtggta ttactgcagc tggtgctata 22620 ccttttgcca cacaactgca ggctagaatt aatcacttgg gtattaccca gtcacttttg 22680 ttgaagaatc aagaaaaaat tgctgcttcc tttaataagg ccattggtcg tatgcaggaa 22740 ggttttagaa gtacatcttt agcattacaa caaattcaag atgttgttaa taagcagagt 22800 gctattctta ctgagactat ggcatcactt aataaaaatt ttggtgccat ttcttctgtg 22860 attcaagaaa tctaccagca acttgacgcc atacaagcaa atgctcaagt ggatcgtctt 22920 ataactggta gattgtcatc actttctgtt ttagcatctg ctaagcaggc ggagtatatt 22980 agagtgtcac aacagcgtga gttagctact cagaagatta atgagtgtgt taagtcacag 23040 tccattaggt actccttttg tggtaatgga cgacatgttt taaccatacc gcaaaatgca 23100 cctaatggta tagtgtttat acacttttct tacactccag atagttttgt taatgttact 23160 gcaatagtgg gtttttgtgt aaagccagct aatgctagtc agtatgcaat agtacccgct 23220 aatggtaggg gtattttcat acaagttaat ggtagttact acatcactgc acgagatatg 23280 tatatgccaa gagctattac tgcaggagat atagttacgc ttacttcttg tcaagtaaat 23340 tatgtaagtg taaataagac cgtcattact acattcgtag acaatgatga ttttgatttt 23400 aatgacgaat tgtcaaaatg gtggaatgat actaagcatg agctaccaga ctttgacaaa 23460 ttcaattaca cagtacctat acttgacatt gatagtgaaa ttgatcgtat tcaaggcgtt 23520 atacagggtc ttaatgactc tctaatagac ctgaaaac ttcaatact caaacttat 23580 attaagtggc cttggtatgt gtggttagcc atagctttg ccactattat cttcatctta 23640 atattaggat gggttttctt catgactggg tgttgtggtt gttgttgttgg atgctttggc 23700 attatgcctc taatgagtaa gtgtggtaag aaatctctt attacacgac tttgataac 23760 gatgtggtaa ctgaacaata cagacctaaa aagtctgttt atgatccaa agtcccacgt 23820 ccttcttaat agtattaatt ttgctttgt gtaaacttgt actaagttgt tttagagagt 23880 ttattatagc gcttcaaca ctaacacaag ttttactcca atttatcgat agtaatttac 23940 agtctagact gaccctttgt cacagtctag actaatgtta aacttagaag cattattga 24000 aactggtgag caagtgattc aaaaaatcag ttcaattta cagcatattt caagtgtatt 24060 aaacacagaa gtatttgacc cctttgacta ttgttac agaggaggta atttttggga 24120 atagagtca gctgaagatt gttcaggtga tgatgaattt attgaatag tcgctagagg 24180 aaaatggaaa ttttctaaca gcgctttaca tatttgtagg atttttagca ctttatcttc 24240 taggtagac acttcaagca tttgtacagg ctgctgatgc ttgttgttta ttttggtata 24300 catgggtagt aattccagga gctaagggta cagcctttgt atacaagtat acatatggta 24360 gaaaacttaa caatccggaa ttagaagcag ttattgtcaa cgagtttcct aagaacggtt 24420 ggaataataa aaatccagca aattttcaag atgtccaacg agacaaattg tactcttgac 24480 tttgaacagt cagttgagct ttttaaagag tataatttat ttataactgc attcttgttg 24540 ttcttaacca tatacttca gtatggctat gcaacaagag gtaagtttat ttatatactg 24600 aaaatgatag tgttatggtg cttttggccc cttaacattg cagtaggtgt aatttcatgt 24660 atatacccac caaacacagg aggtcttgtc gcagcgataa tacttacagt gtttgcgtgt 24720 ctgtctttg taggttattg gatccagagt attagactct ttaagcggtg taggtcatgg 24780 tggtcattta acccagaatc taatgccgta ggttcaatac tcctaactaa tggtcaacaa 24840 tgtaatttg ctatagagag tgtgccaatg gtgctttctc caattataaa gaatggtgtt 24900 ctttattgtg agggtcagtg gcttgctaag tgtgaaccag accacttgcc taaagatata 24960 tttgtttgta caccggatag acgtaatatc taccgtatgg tgcagaaata tactggtgac 25020 caaagcggaa atagaaag gtttgctacg ttgtctatg caaagcagtc agtagacact 25080 ggcgagctag aaagtgtagc aacaggtgga agtagccttt acacataaat gtgtgtgtgt 25140 agaggagtatt taaaattatt cttcatagt gcctctttt taagagcgcg gaagagtatt 25200 tgttttgagg atattatat aaatccctt tgttttgtac tctctttaca agagttatta 25260 tttaagcaac agttttcct ttccttgtt tggaagaaag ttgttgttaa tggtgtagaa 25320 ttccaagtag aaaatggaaa agtccactac gaaggaaacc ccattctcta aaaaggttgt 25380 tgtaggttgt gtcccatta taagagat taatggatt aaaccaccta cactacttac 25440 ttgtataag ggcgtttgga cttacaagcg cttaacaaat acagacgatg aaatggctga 25500 ctagttttgg agagcagtt atttcttgtt ataaagccct actattaact cagttaagg 25560 tattagatag gttaatttta gatcacggac caaaacgcgt cttaacgtgt ggtaggcgag 25620 tgcttttatc tcaattagat ctagtttata ggttggcata tacgcccacc caatcgctgg 25680 tatgaataat agtaaagata atccttttcg cggagcaata gcaagaaaag cgcgaattta 25740 tctgagagaa ggattagagt gtgtttactt tcttaacaaa gcaggacaag cagagtcttg 25800 tcccgcgtg...

Claims

1. An infectious bronchitis virus H52, encoding a heterologous infectious bronchitis virus S protein, wherein the heterologous infectious bronchitis virus S protein consists of one of the following amino acid sequences: the amino acid sequences shown in SEQ ID NO: 5, 6, 7 and 8.

2. The infectious bronchitis virus H52 of claim 1, wherein the infectious bronchitis virus H52 is attenuated or recombinant.

3. An immunogenic composition comprising the infectious bronchitis virus H52 of any one of claims 1-2.

4. The immunogenic composition of claim 3, wherein the immunogenic composition is a vaccine.

5. A kit comprising the infectious bronchitis virus H52 of any one of claims 1-2, or the immunogenic composition of any one of claims 3-4.

6. Use of the infectious bronchitis virus H52 according to any one of claims 1 to 2 for the preparation of an immunogenic composition for treating or preventing an infection caused by an infectious bronchitis virus in poultry in need thereof.

7. Use of the infectious bronchitis virus H52 according to any one of claims 1 to 2 for the preparation of an immunogenic composition for reducing infectious bronchitis virus-induced ciliary arrest in poultry in need thereof compared to a non-immunized control group of the same poultry.

8. The use of any one of claims 6-7, wherein the immunogenic composition results in an improvement in an efficacy parameter selected from the group consisting of prevention or reduction of infectious bronchitis virus-induced ciliary arrest, prevention or reduction of rales, prevention or reduction of egg drop, prevention or reduction of kidney lesions, prevention or reduction of watery diarrhea, prevention or reduction of weight loss, reduction of infectious bronchitis virus load, reduction of infectious bronchitis virus shedding, or a combination thereof, compared to an untreated control group of the same poultry.

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