4 / 91 IBV vaccine with heterologous spike protein

Abstract

The present invention relates to 4 / 91 IBV (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 4 / 91 IBV encoding a 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 to the subject a therapeutically effective amount of an immunogenic composition according to the present invention.

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 4 / 91 IBV (Infectious Bronchitis Virus) encoding a heterologous IBV S (Spike) protein or a fragment thereof.

[0017] The term "4 / 91 IBV" is well known to those skilled in the art. The term "IBV" refers to infectious bronchitis virus. The term "4 / 91" is used interchangeably with the term "793B" to define a specific IBV serotype or genotype. The 4 / 91 serotype or genotype is well known to those skilled in the art and includes strains such as UK / 4 / 91, UK / 793B / 91, and CR88. 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] How to obtain 4 / 91 IBV is common knowledge among those skilled in the art. 4 / 91 IBV strains are commercially available, for example IB 4-91 (MSD Animal Health), CEVAC (CEVA; strain 1 / 96), or Gallivac IB88 (Boehringer Ingelheim; strain CR88121). Furthermore, the 4 / 91 IBV-specific vaccine strain has been used for decades (Jones 2010 Rev. Bras. Cienc. Avic. vol. 12 no. 2) and can therefore be isolated in the field. Methods for isolating and identifying 4 / 91 IBV strains are well known to those skilled in the art. For example, the identification of the 4 / 91 IBV strain can be as described in SHIMAZAKI et al. 2008 (J. Vet. Med. Sci. 71 (5): 583-588), Martin et al. 2014 (Avian Pathology Vol. 43, No. 3, 264-268), Zanaty et al. 2016 (World J Virol; 5 (3): 125-134), or Stenzel et al. 2017 (Polish Journal of Veterinary Sciences Vol. 20, No. 3, 599-601). In addition, 4 / 91 IBV has been sequenced and a genome sequence is available, such as KF377577. Therefore, the viral genome can be generated by synthesizing its sequence and can be generated by applying a reverse genetics system.

[0019] 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.

[0020] The term "heterologous S (spike protein)" refers to a spike protein or fragment thereof that has been introduced into 4 / 91 IBV that is from a different genotype or serotype than 4 / 91 IBV. Thus, the heterologous spike protein is a non-4 / 91 genotype or serotype.

[0021] 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.

[0022] Furthermore, the present invention also provides an immunogenic composition comprising 4 / 91 IBV (infectious bronchitis virus) encoding a heterologous S (spike) protein or a fragment thereof.

[0023] 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 4 / 91 IBV (Infectious Bronchitis Virus) encoding a heterologous S (Spike) protein or a fragment thereof.

[0024] 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.

[0025] 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.

[0026] 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.

[0027] 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.

[0028] 4 / 91-IBV-defined by protein coding sequence

[0029] In another specific aspect of the IBV or immunogenic composition of the present invention, the 4 / 91 IBV has a nucleotide sequence as shown in KF377577 (SEQ ID NO:53) 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.

[0030] In another specific aspect of the IBV or immunogenic composition of the present invention, the 4 / 91 IBV has a nucleotide sequence as shown in SEQ ID NO: 1 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.

[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 present invention, the 4 / 91 IBV strain has a spike (S) protein having an amino acid sequence as shown in AGY56140 (SEQ ID NO:54) 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.

[0034] In another specific aspect of the IBV or immunogenic composition of the present invention, the 4 / 91 IBV strain has a spike (S1) protein, and the spike protein S1 has the amino acid sequence shown in AF093793 (SEQ ID NO:60) 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.

[0035] In another specific aspect of the IBV or immunogenic composition of the present invention, the 4 / 91 IBV strain has a spike (S1) protein, and the spike protein S1 has an amino acid sequence as shown in EU914938 (SEQ ID NO: 55) 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.

[0036] In another specific aspect of the IBV or immunogenic composition of the present invention, the 4 / 91 IBV strain has a spike (S1) protein, and the spike protein S1 has an amino acid sequence as shown in KM067900 (SEQ ID NO: 56) 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.

[0037] It must be understood that the spike protein sequence or nucleic acid can be used to determine whether any IBV strain is derived from 4 / 91. However, since the 4 / 91 IBV is used as a backbone and the 4 / 91 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 4 / 91 spike protein or only a portion of the 4 / 91 spike protein remains.

[0038] In another specific aspect of the IBV or immunogenic composition of the present invention, the 4 / 91 IBV strain has a spike (S) protein having an amino acid sequence as shown in SEQ ID NO: 2 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.

[0039] In another specific aspect of the IBV or immunogenic composition of the present invention, the 4 / 91 IBV strain has a nucleocapsid (N) protein having an amino acid sequence as shown in EU780081 (SEQ ID NO: 57) 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 with at least one of the above sequences.

[0040] In another specific aspect of the IBV or immunogenic composition of the present invention, the 4 / 91 IBV strain has a nucleocapsid (N) protein having an amino acid sequence as 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 4 / 91 IBV strain has an envelope (E) protein having an amino acid sequence as shown in AGY56143 (SEQ ID NO:58) 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.

[0042] In another specific aspect of the IBV or immunogenic composition of the present invention, the 4 / 91 IBV strain has an envelope (E) protein 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] In another specific aspect of the IBV or immunogenic composition of the present invention, the 4 / 91 IBV strain has a membrane glycoprotein (M) having an amino acid sequence as shown in AGY56144 (SEQ ID NO: 59) 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.

[0044] In another specific aspect of the IBV or immunogenic composition of the present invention, the 4 / 91 IBV strain has a membrane glycoprotein (M), which has an amino acid sequence as shown in SEQ ID NO:5 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.

[0045] 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.

[0046] 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.

[0047] 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.

[0048] 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.

[0049] 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 / .

[0050] 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 1-61, and thus "SEQ ID NO: X" represents any SEQ ID NO mentioned herein.

[0051] According to another specific aspect of the IBV or immunogenic composition of the present invention, the 4 / 91 IBV is selected from the following group of strains: 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 / 4 / 91, UK / 7 / 91, UK / 793B / 91, 4 / 91-pathogenic, 4 / 91 attenuated, IB4-91 and CR88.

[0052] Heterologous S protein

[0053] IBV strains

[0054] 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, those skilled in the art are familiar with the different IBV strains. Infectious bronchitis virus was first discovered in the United States in the 1930s.

[0055] The first IBV serotype was identified in Massachusetts and remained the only serotype until a different IBV serotype was discovered in 1956. Today, in addition to the originally identified Massachusetts serotype, several other serotypes have been identified in the United States, including Arkansas and Delaware. Today, IBV Mass viruses can be identified in many countries around the world.

[0056] 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.

[0057] 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.

[0058] 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.

[0059] 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.

[0060] In addition, strains D274, B1648 / D8880, D1466, V1397 and Arkansas have also been identified in Europe.

[0061] 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.

[0062] In another specific aspect of the IBV or immunogenic composition of the invention, the heterologous spike protein is of a non-4 / 91 genotype or serotype.

[0063] 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), Massachusetts (M41, H52, H120), Pennsylvania (e.g., Pennsylvania1220 / 98, Pennsylvania Wolg / 98), PL84084, Qu (e.g., Qu-mv), QX (e.g., GB341 / 96), Q1, SE 17, and variant 2 (e.g., IS / 1494 / 06, IBV / Ck / EG / CU / 4 / 2014, gammaCoV / Ck / Poland / G052 / 2016).

[0064] 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: Massachusetts, QX, Q1, Italy 02, Arkansas, Conneticut, Georgia, LDT3, PL84084, variant 2 and Brazil.

[0065] 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 the group consisting of Massachusetts, QX, Q1, Arkansas, variant 2 and Brazil.

[0066] According to another specific aspect of the IBV or immunogenic composition of the present invention, the Massachusetts strain is selected from the group consisting of H120, H52, Spain / 98 / 308, IBMA5-1, SD / 97 / 01, Spain / 96 / 334, M41-M21883.

[0067] 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.

[0068] 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.

[0069] 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.

[0070] 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.

[0071] 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.

[0072] 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 Massachusetts genotype or serotype.

[0073] In another specific aspect of the IBV or immunogenic composition of the invention, the heterologous S protein or fragment thereof is from the Massachusetts 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:6 or 7.

[0074] 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: 8 or 9.

[0075] 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: 10 or 11.

[0076] 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:12 or 13.

[0077] 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: 14 or 15.

[0078] 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:16 or 17.

[0079] 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: 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17.

[0080] In another specific aspect of the IBV or immunogenic composition of the invention, the heterologous S protein is a full-length spike protein.

[0081] The experimental data of the present invention indicate that the full-length spike protein sequence can be used. However, it is known that fragments of the spike protein sequence and the extracellular domain of the spike protein can also be used.

[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 500, 750, 1000 or 1075 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 at least 500, 750, 1000 or 1075 amino acids in length from the N-terminus.

[0084] 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).

[0085] 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.

[0086] 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.

[0087] The term "ectodomain" is well known to those skilled in the art. The spike protein comprises different 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 at the extracellular domain. The length of the IBV spike protein extracellular domain is approximately 1075 amino acids, varying by a few amino acids depending on the IBV strain.

[0088] 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.

[0089] 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.

[0090] 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 4 / 91 IBV.

[0091] In another specific aspect of the IBV or immunogenic composition of the invention, the IBV is attenuated.

[0092] 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.

[0093] In another specific aspect of the IBV or immunogenic composition of the invention, the IBV is inactivated.

[0094] 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.

[0095] 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.

[0096] Preferably, the inactivated IBV of the present invention is formalin-inactivated, preferably using the concentrations described herein above.

[0097] 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.

[0098] In another specific aspect of the IBV or immunogenic composition of the invention, the IBV is genetically engineered.

[0099] 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.

[0100] In another specific aspect of the IBV or immunogenic composition of the invention, the IBV is a recombinant IBV.

[0101] 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.

[0102] In another specific aspect of the IBV or immunogenic composition of the invention, the IBV is chimeric.

[0103] 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 4 / 91 encoding a heterologous S (spike) protein or a fragment thereof is a chimeric IBV.

[0104] 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."

[0105] In another specific aspect of the immunogenic composition according to the present invention, the immunogenic composition comprises a pharmaceutically acceptable carrier.

[0106] 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.

[0107] "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.

[0108] In another specific aspect of the immunogenic composition of the present invention, the pharmaceutically acceptable carrier is phosphate buffered saline.

[0109] Preferably, the immunogenic composition further comprises a sucrose gelatin stabilizer.

[0110] Preferably, the pharmaceutically acceptable carrier is chitosan.

[0111] Chitosan is a natural deacetylated polysaccharide derived from chitin in crustaceans (such as shrimp and crab), 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.

[0112] 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.

[0113] 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.

[0114] 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.

[0115] 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.

[0116] 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%.

[0117] 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.

[0118] 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.

[0119] In another specific aspect of the immunogenic composition according to the present invention, the immunogenic composition protects against challenge with a Massachusetts, QX, Q1, Arkansas, variant 2 or Brazil genotype strain.

[0120] In another specific aspect of the immunogenic composition according to the present invention, the immunogenic composition protects against challenge with a Massachusetts genotype strain.

[0121] In another specific aspect of the immunogenic composition according to the present invention, the immunogenic composition is formulated for single-dose administration.

[0122] The volume of a single dose has been defined elsewhere herein.

[0123] 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.

[0124] 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.

[0125] 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.

[0126] 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.

[0127] 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.

[0128] Reagent test kit

[0129] 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.

[0130] Thus, the present invention provides a kit comprising an IBV or immunogenic composition as described herein.

[0131] 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.

[0132] In a specific aspect of the kit according to the present invention, the kit further comprises instructions for treating and / or preventing poultry diseases.

[0133] 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).

[0134] Treatment

[0135] Furthermore, the present invention provides methods for immunizing a subject comprising administering to the subject an immunogenic composition as described herein.

[0136] 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.

[0137] 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.

[0138] 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.

[0139] 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.

[0140] 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.

[0141] 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.

[0142] "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.

[0143] 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.

[0144] 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.

[0145] 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.

[0146] 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.

[0147] 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.

[0148] 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.

[0149] 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.

[0150] As shown in the Examples, the immunogenic compositions provided herein have been demonstrated to be effective in reducing ciliary arrest.

[0151] 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.

[0152] 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.

[0153] 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.

[0154] In one aspect of the invention, the subject is an avian.

[0155] The term "avian" is well known to those skilled in the art. The term "avian" includes all birds, including poultry.

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

[0157] 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.

[0158] In one aspect of the invention, the subject is selected from the group consisting of chicken, turkey, quail or pheasant.

[0159] In one aspect of the invention, the subject is a chicken.

[0160] In one aspect of the invention, the immunogenic composition is administered once.

[0161] 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.

[0162] The dose volume per bird depends on the route of administration and the age of the bird.

[0163] 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.

[0164] 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.

[0165] 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.

[0166] 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.

[0167] In one aspect of the invention, the immunogenic composition is administered in two or more doses.

[0168] However, the immunogenic composition may be administered in two or more doses, with a first dose being administered before a second dose (boost dose).

[0169] 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.

[0170] 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.

[0171] 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.

[0172] 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.

[0173] 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.

[0174] The live IBV vaccine is preferably administered alone via eye drops, intranasally, intramuscularly or subcutaneously.

[0175] 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.

[0176] 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.

[0177] In ovo administration

[0178] 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.

[0179] 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.

[0180] 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.

[0181] In one aspect of the invention, the immunogenic composition is administered by eye drops.

[0182] 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 unit dose 2 to 10 4 EID 50 , even more preferably 10 per dose 2 to 10 3 EID 50 .

[0183] 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.

[0184] 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 / ml, preferably 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 .

[0185] In one aspect of the invention, the immunogenic composition comprises 1 to 10 log 10 IBV of EID50.

[0186] In one aspect of the invention, the immunogenic composition comprises 2 to 5 log 10 IBV of EID50.

[0187] In one aspect of the invention, the immunogenic composition comprises 2-4 log 10 IBV of EID50.

[0188] 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.

[0189] 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.

[0190] 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.

[0191] 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.

[0192] 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.

[0193] 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.

[0194] 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.

[0195] 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.

[0196] 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.

[0197] 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.

[0198] The term "egg drop" is well known to those skilled in the art. The term "egg drop" includes a reduction in egg production.

[0199] 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.

[0200] 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.

[0201] 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.

[0202] The present invention further provides an IBV or immunogenic composition as described herein for use in therapeutic treatment.

[0203] The present invention further provides an IBV or immunogenic composition as described herein for use as an immunogen or vaccine.

[0204] The present invention also provides an IBV or immunogenic composition as described herein for use as a medicament.

[0205] The present invention further provides use of the IBV or immunogenic composition described herein in the preparation of a medicament.

[0206] 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.

[0207] The present invention further provides an immunogenic composition comprising a 4 / 91 IBV (infectious bronchitis virus) encoding a heterologous S (spike) protein or a fragment thereof, wherein the 4 / 91 IBV comprises a nucleocapsid (N) protein, an envelope (E) protein or a membrane glycoprotein (M) having a sequence such as SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, EU780081 (SEQ ID NO 57), AGY56143 (SEQ ID NO 58), AGY56144 (SEQ ID NO 59) 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 genotypes or serotypes of Massachusetts, QX, Q1, Arkansas, variant 2 and Brazil, or is selected from the genotypes or serotypes of SEQ ID The amino acid sequence shown in NO:6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17, 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.

[0208] In another specific aspect of the immunogenic composition according to the present invention, the heterologous S protein is a full-length spike protein.

[0209] 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 1075 amino acids starting from the N-terminus.

[0210] 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.

[0211] In another specific aspect of the immunogenic composition according to the invention, the IBV is attenuated.

[0212] In another specific aspect of the immunogenic composition according to the present invention, the 4 / 91 IBV is selected from the genotype or serotype of CR88 and 793B.

[0213] 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:

[0214] a.) providing a 4 / 91 IBV comprising a spike (S) protein, a nucleocapsid (N) protein, an envelope (E) protein or a membrane glycoprotein (M) having a sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, AGY6140 (SEQ ID NO 54), AF093793 (SEQ ID NO 60), EU914938 (SEQ ID NO 55), KM067900 (SEQ ID NO 56), EU780081 (SEQ ID NO 57), AGY56143 (SEQ ID NO 58), AGY56144 (SEQ ID NO 59), 59), 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

[0215] b.) providing a heterologous S protein or fragment thereof selected from the list of genotypes or serotypes of Massachusetts, QX, Q1, Arkansas, variant 2, and Brazil, or selected from the amino acid sequence of SEQ ID NO: 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17, 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

[0216] c.) replacing the spike protein or fragment thereof of the 4 / 91 IBV in a) with the heterologous S (spike) protein or fragment thereof described in b) to obtain a 4 / 91 IBV having a heterologous S protein or fragment thereof; and

[0217] d.) obtaining the 4 / 91 IBV having a heterologous S protein or a fragment thereof; and

[0218] e.) Adding a pharmaceutically acceptable carrier.

[0219] The term "obtaining" includes harvesting, separation, purification and / or formulation (eg, completion, inactivation and / or mixing) of said IBV 4 / 91 with a heterologous S protein or a fragment thereof.

[0220] The term "harvesting" refers to collecting or recovering the IBV 4 / 91 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.

[0221] The term "isolation" includes the step of isolating the IBV 4 / 91 containing the heterologous S protein or fragment thereof. Methods for isolating the IBV 4 / 91 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.

[0222] Methods for "purifying" the IBV 4 / 91 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.

[0223] 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.

[0224] 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.

[0225] 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.

[0226] 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.

[0227] In another specific aspect of the method for preparing an immunogenic composition according to the invention, the 4 / 91 IBV is selected from the list of strains 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 / 4 / 91, UK / 7 / 91, UK / 793B / 91, 4 / 91-pathogenic, 4 / 91 attenuated, IB4-91 and CR88.

[0228] The present invention further relates to a plasmid comprising a nucleic acid encoding a portion of the 4 / 91 IBV (Infectious Bronchitis Virus) genome, said genome including a heterologous IBV S (Spike) protein or a fragment thereof, such as the pUC57-S CR88 rIBVH52 S donor plasmid (SEQ ID NO: 21).

[0229] Terms

[0230] This article also describes the following terms:

[0231] 1. 4 / 91 IBV (Infectious Bronchitis Virus) encoding a heterologous IBV S (Spike) protein or a fragment thereof.

[0232] 2. An immunogenic composition comprising 4 / 91 IBV (Infectious Bronchitis Virus) encoding a heterologous S (Spike) protein or a fragment thereof.

[0233] 3. An immunogenic composition comprising IBV (Infectious Bronchitis Virus) according to clause 1.

[0234] 4 / 91-IBV-Protein Coding Sequence Definition

[0235] 4. The IBV or immunogenic composition of any one of clauses 1 to 3, wherein the 4 / 91 IBV has, consists of, or comprises the nucleotide sequence shown as KF377577 (SEQ ID NO 53), 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.

[0236] 5. The IBV or immunogenic composition of any one of clauses 1 to 4, wherein the 4 / 91 IBV has, consists of, or comprises the nucleotide sequence of SEQ ID NO 1 or a sequence having at least 90%, 91%, 92%, 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.

[0237] 6. IBV or the immunogenic composition of any one of clauses 1 to 5, wherein the 4 / 91 IBV strain has, consists of, or comprises a spike protein having the amino acid sequence of AGY56140 (SEQ ID NO 54) 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.

[0238] 7. The IBV or immunogenic composition of any one of clauses 1 to 6, wherein the 4 / 91 IBV strain has, consists of or comprises a Spike (S1) protein having the amino acid sequence of AF093793 (SEQ ID NO 60) 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.

[0239] 8. The IBV or immunogenic composition of any one of clauses 1 to 7, wherein the 4 / 91 IBV strain has, consists of or comprises a Spike (S1) protein having the amino acid sequence of EU914938 (SEQ ID NO 55) 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.

[0240] 9. The IBV or immunogenic composition of any one of clauses 1 to 8, wherein the 4 / 91 IBV strain has, consists of, or comprises a Spike (S1) protein having the amino acid sequence of KM067900 (SEQ ID NO 56) 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.

[0241] 10. The IBV or immunogenic composition of any one of clauses 1 to 9, wherein the 4 / 91 IBV strain has, consists of, or comprises a spike (S) protein having the amino acid sequence of SEQ ID NO: 2 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.

[0242] 11. The IBV or immunogenic composition of any one of clauses 1 to 10, wherein the 4 / 91 IBV has, consists of or comprises a nucleocapsid (N) protein having the amino acid sequence of EU780081 (SEQ ID NO 57) 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.

[0243] 12. The IBV or immunogenic composition of any one of clauses 1 to 11, wherein the 4 / 91 IBV has, consists of, or comprises a nucleocapsid (N) 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.

[0244] 13. The IBV or immunogenic composition of any one of clauses 1 to 12, wherein 4 / 91 IBV has, consists of or comprises an envelope (E) protein having the amino acid sequence of AGY56143 (SEQ ID NO 58) 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.

[0245] 14. The IBV or immunogenic composition of any one of clauses 1 to 13, wherein 4 / 91 IBV has, consists of, or comprises an envelope (E) 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.

[0246] 15. The IBV or immunogenic composition of any one of clauses 1 to 14, wherein 4 / 91 IBV has, consists of or comprises a membrane glycoprotein (M) having the amino acid sequence of AGY56144 (SEQ ID NO 59) 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.

[0247] 16. The IBV or immunogenic composition of any one of clauses 1 to 15, wherein 4 / 91 IBV has, consists of, or comprises a membrane glycoprotein (M) protein having the amino acid sequence of SEQ ID NO: 5, 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.

[0248] 17. The IBV or immunogenic composition of any one of clauses 1 to 16, wherein 4 / 91 IBV 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 / 4 / 91, UK / 7 / 91, UK / 793B / 91, 4 / 91-pathogenic, 4 / 91-attenuated, IB4-91 and CR88.

[0249] Heterologous S protein

[0250] 18. The IBV or immunogenic composition of any one of clauses 1 to 17, wherein the heterologous spike is of a non-4 / 91 genotype or serotype.

[0251] 19. The IBV or immunogenic composition of any one of clauses 1 to 18, 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), Massachusetts (M41, H52, H120), Pennsylvania (e.g., Pennsylvania1220 / 98, Pennsylvania Wolg / 98), PL84084, Qu (e.g., Qu-mv), QX (e.g., GB341 / 96), Q1, SE 17, and variant 2 (e.g., IS / 1494 / 06, IBV / Ck / EG / CU / 4 / 2014, gammaCoV / Ck / Poland / G052 / 2016).

[0252] 20. The IBV or immunogenic composition of any one of clauses 1 to 19, wherein the heterologous S protein or fragment thereof is from an IBV genotype or serotype selected from the group consisting of Massachusetts, QX, Q1, Italy 02, Arkansas, Conneticut, Georgia, LDT3, PL84084, variant 2 or Brazil.

[0253] 21. The IBV or immunogenic composition of any one of clauses 1 to 20, wherein the heterologous S protein or fragment thereof is from an IBV of a genotype or serotype selected from the group consisting of: Massachusetts, QX, Q1, Arkansas, variant 2, and Brazil.

[0254] 22. The IBV or immunogenic composition of clause 21 wherein the Massachusetts strain is selected from the group consisting of: H120, H52, Spain / 98 / 308, IBMA5-1, SD / 97 / 01, Spain / 96 / 334, M41-M21883.

[0255] 23. The IBV or immunogenic composition of clause 21, 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.

[0256] 24. The IBV or immunogenic composition of clause 21, 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.

[0257] 25. The IBV or immunogenic composition of clause 21 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.

[0258] 26. The IBV or immunogenic composition of clause 21 , 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.

[0259] 27. The IBV or immunogenic composition of clause 21, wherein the Brazil strain is selected from BR-1, BR-2, 23 / 2013 and IBV / Brasil / 351 / 1984.

[0260] 28. The IBV or immunogenic composition of any one of clauses 1 to 27, wherein the heterologous S protein or fragment thereof is from the Massachusetts genotype or serotype.

[0261] 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 Massachusetts 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: 6 or 7, or the heterologous S protein or fragment thereof comprises or consists of SEQ ID NO: 6 or 7. NO:6 or 7, 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.

[0262] 30. The IBV or immunogenic composition of any one of clauses 1 to 29, 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: 8 or 9, or the heterologous S protein or fragment thereof comprises or consists of SEQ ID NO: 9. The invention also comprises the amino acid sequence shown in NO:8 or 9, 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.

[0263] 31. The IBV or immunogenic composition of any one of clauses 1 to 30, 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: 10 or 11, 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:10 or 11, 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.

[0264] 32. The IBV or immunogenic composition of any one of clauses 1 to 31 , 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: 12 or 13, or the heterologous S protein or fragment thereof comprises or consists of SEQ ID NO: The amino acid sequence of NO:12 or 13, 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.

[0265] 33. The IBV or immunogenic composition of any one of clauses 1 to 32, 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: 14 or 15, 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:14 or 15, 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.

[0266] 34. The IBV or immunogenic composition of any one of clauses 1 to 33, 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: 16 or 17, or the heterologous S protein or fragment thereof comprises or consists of SEQ ID NO: The amino acid sequence of NO:16 or 17, 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.

[0267] 35. The IBV or immunogenic composition of any one of clauses 1 to 34, wherein the heterologous S protein or fragment thereof is selected from SEQ ID NO: 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17.

[0268] 36. The IBV or immunogenic composition of any one of clauses 1 to 35, wherein the heterologous S protein is a full-length spike protein.

[0269] 37. The IBV or immunogenic composition of any one of clauses 1 to 36, wherein the fragment of the heterologous S (Spike) protein is at least 500, 750, 1000 or 1075 amino acids in length.

[0270] 38. The IBV or immunogenic composition of any one of clauses 1 to 37, wherein the fragment of the heterologous S (Spike) protein is at least 500, 750, 1000 or 1075 amino acids in length from the N-terminus.

[0271] 39. The IBV or immunogenic composition of any one of clauses 1 to 38, wherein the fragment of the heterologous S (Spike) protein is at least 1000 amino acids in length.

[0272] 40. The IBV or immunogenic composition of any one of clauses 1 to 39, wherein the heterologous S (Spike) protein is the extracellular domain of the Spike protein.

[0273] 41. The IBV or immunogenic composition of any one of clauses 1 to 40, wherein the heterologous S (Spike) protein or fragment thereof replaces the homologous S protein or fragment thereof.

[0274] 42. The IBV or immunogenic composition of any one of clauses 1 to 41 , wherein the heterologous S (Spike) protein or fragment thereof replaces a naturally occurring S protein or fragment thereof.

[0275] 43. The IBV or immunogenic composition of any one of clauses 1 to 42, wherein the heterologous S (Spike) protein or fragment thereof replaces 4 / 91 of the S protein or fragment thereof.

[0276] 44. The IBV or immunogenic composition of any one of clauses 1 to 43, wherein the IBV is attenuated.

[0277] 45. The IBV or immunogenic composition of any one of clauses 1 to 43, wherein the IBV is inactivated.

[0278] 46. ​​The IBV or immunogenic composition of any one of clauses 1 to 45, wherein the IBV is genetically engineered.

[0279] 47. The IBV or immunogenic composition of any one of clauses 1 to 46, wherein the IBV is a recombinant IBV.

[0280] 48. The immunogenic composition of any one of clauses 2 to 47 wherein the immunogenic composition is a vaccine.

[0281] 49. The immunogenic composition of any one of clauses 2 to 48, wherein the immunogenic composition comprises a pharmaceutically acceptable carrier.

[0282] 50. The immunogenic composition of clause 49, wherein the pharmaceutically acceptable carrier is phosphate buffered saline.

[0283] 51. The immunogenic composition of any one of clauses 2 to 50, wherein the immunogenic composition is effective in treating and / or preventing clinical signs caused by IBV in a subject in need thereof.

[0284] 52. The immunogenic composition of any one of clauses 2 to 51 , wherein the immunogenic composition protects against challenge with an IBV strain having a genotype or serotype of a heterologous spike protein.

[0285] 53. The immunogenic composition of any one of clauses 2 to 52, wherein the immunogenic composition protects against challenge with a Massachusetts, QX, Q1, Arkansas, variant 2 or Brazil genotype strain.

[0286] 54. The immunogenic composition of any one of clauses 2 to 53 wherein the immunogenic composition protects against challenge with a Massachusetts genotype strain.

[0287] 55. The immunogenic composition of any one of clauses 2 to 54, wherein the immunogenic composition is formulated for single dose administration.

[0288] 56. The immunogenic composition of any one of clauses 2 to 55 wherein the immunogenic composition is administered subcutaneously, intramuscularly, orally, in ovo, by spray, by drinking water or as eye drops.

[0289] 57. The immunogenic composition of any one of clauses 2 to 56, wherein the immunogenic composition comprises 1 to 10 log 10 EID 50 / dose of IBV.

[0290] 58. The immunogenic composition of any one of clauses 2 to 57, wherein the immunogenic composition comprises 2 to 5 log 10 EID 50 / dose of IBV.

[0291] 59. The immunogenic composition of any one of clauses 2 to 58, wherein the immunogenic composition comprises 2 to 4 log 10 EID 50 / dose of IBV.

[0292] 60. A kit comprising the IBV or immunogenic composition of any one of clauses 1-59.

[0293] 61. The kit according to clause 60, wherein the kit further comprises instructions for use in treating and / or preventing avian diseases.

[0294] 62. The kit according to clause 60, wherein the kit further comprises instructions for use in treating and / or preventing poultry diseases.

[0295] 63. The kit according to clause 60, wherein the kit further comprises instructions for use in treating and / or preventing IB.

[0296] 64. A method of immunizing a subject comprising administering to the subject an immunogenic composition according to any one of clauses 2 to 59.

[0297] 65. 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 59.

[0298] 66. 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 to 59.

[0299] 67. The immunogenic composition of any one of clauses 2 to 59 for use in a method of immunizing a subject, the method comprising administering to the subject a therapeutically effective amount of the immunogenic composition.

[0300] 68. The immunogenic composition of any one of clauses 2 to 59 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.

[0301] 69. The immunogenic composition of any one of clauses 2 to 59 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.

[0302] 70. The method or use of any one of clauses 64 to 69, wherein the subject is a poultry.

[0303] 71. The method or use of any one of clauses 64 to 70 wherein the subject is poultry.

[0304] 72. The method or use of any one of clauses 64 to 71 wherein the subject is selected from a chicken, a turkey, a quail or a pheasant.

[0305] 73. The method or use of any one of clauses 64 to 72 wherein the subject is a chicken.

[0306] 74. The method or use of any one of clauses 64 to 73 wherein the immunogenic composition is administered once.

[0307] 75. The method or use of any one of clauses 64 to 73 wherein the immunogenic composition is administered in two or more doses.

[0308] 76. The method or use of any one of clauses 64 to 75 wherein the immunogenic composition is administered subcutaneously, intramuscularly, orally, by in ovo injection, by spray, by drinking water or as eye drops.

[0309] 77. The method or use of any one of clauses 64 to 76 wherein the immunogenic composition is administered by eye drops.

[0310] 78. The method or use of any one of clauses 64 to 77, wherein the immunogenic composition comprises 1 to 10 log 10 EID 50 / dose of IBV.

[0311] 79. The method or use of any one of clauses 64 to 78, wherein the immunogenic composition comprises 2 to 5 log 10 EID 50 / dose of IBV.

[0312] 80. The method or use of any one of clauses 64 to 79, wherein the immunogenic composition comprises 2 to 4 log 10 EID 50 / dose of IBV.

[0313] 81. The method or use of any one of clauses 64 to 80, 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.

[0314] 82. The method or use of any one of clauses 64 to 81 wherein the immunogenic composition is administered to the subject within the first day of the subject's age.

[0315] 83. The method or use of any one of clauses 64 to 82, 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 pathology, 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.

[0316] 84. The method or use of any one of clauses 64 to 83, wherein the treating or preventing results in prevention or reduction of ciliary arrest compared to an untreated control subject of the same species.

[0317] 85. The method or use of any one of clauses 64 to 84, wherein the treating or preventing results in prevention or reduction of kidney pathology compared to an untreated control subject of the same species.

[0318] 86. The method or use of any one of clauses 64 to 85, 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.

[0319] 87. The IBV or immunogenic composition of any one of clauses 1 to 59 for use in therapy.

[0320] 88. The IBV or immunogenic composition of any one of clauses 1 to 59 for use as an immunogen or vaccine.

[0321] 89. The IBV or immunogenic composition of any one of clauses 1 to 59 for use as a medicament.

[0322] 90. Use of the IBV or immunogenic composition of any one of clauses 1 to 59 in the manufacture of a medicament.

[0323] 91. Use of the IBV or immunogenic composition of any one of clauses 1 to 59 for treating and / or preventing IBV infection in a subject.

[0324] 92. An immunogenic composition comprising a 4 / 91 IBV (infectious bronchitis virus) encoding a heterologous S (spike) protein or a fragment thereof, wherein the 4 / 91 IBV comprises a nucleocapsid (N) protein, an envelope (E) protein or a membrane glycoprotein (M) having or comprising or consisting of the following sequences: SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, EU780081 (SEQ ID NO: 57), AGY56143 (SEQ ID NO: 58), AGY56144 (SEQ ID NO:59) 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 following genotypes or serotypes: Massachusetts, QX, Q1, Arkansas, variant 2 and Brazil, or is selected from the following amino acid sequences: SEQ ID ... NO:6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17, 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 a fragment thereof comprising or consisting of the following amino acid sequence: SEQ ID NO:6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17, 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: 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17, 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.

[0325] 93. The immunogenic composition of clause 92, wherein the heterologous S protein is a full-length spike protein.

[0326] 94. The immunogenic composition of clause 92 or 93, wherein the fragment of the heterologous S (spike) protein is at least 500, 750, 1000 or 1075 amino acids in length from the N-terminus.

[0327] 95. The immunogenic composition of any one of clauses 92 to 94, wherein the fragment of the heterologous S (Spike) protein is the extracellular domain of the Spike protein.

[0328] 96. The immunogenic composition of any one of clauses 92 to 95 wherein the IBV is attenuated.

[0329] 97. The immunogenic composition of any one of clauses 92 to 96 wherein 4 / 91 IBV 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 / 4 / 91, UK / 7 / 91, UK / 793B / 91, 4 / 91-pathogenic, 4 / 91-attenuated, IB4-91 and CR88.

[0330] 98. A method of preparing an immunogenic composition for treating and / or preventing IBV infection in a subject, comprising:

[0331] a.) providing a 4 / 91 IBV comprising a spike (S) protein, a nucleocapsid (N) protein, an envelope (E) protein or a membrane glycoprotein (M) having or comprising or consisting of the following amino acid sequence: SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, AGY6140 (SEQ ID NO: 54), AF093793 (SEQ ID NO: 60), EU914938 (SEQ ID NO: 55), KM067900 (SEQ ID NO: 56), EU780081 (SEQ ID NO: 57), AGY56143 (SEQ ID NO: 58), AGY56144 (SEQ ID NO: 59), AF093793 (SEQ ID NO: 61), EU914938 (SEQ ID NO: 62), KM067900 (SEQ ID NO: 63), EU780081 (SEQ ID NO: 64), AGY56145 (SEQ ID NO: 65), AGY56146 (SEQ ID NO: 66), AGY56147 (SEQ ID NO: 67), AGY56148 (SEQ ID NO: 68), AGY56149 (SEQ ID NO: 69), AGY56149 (SEQ ID NO: 70), AGY56141 (SEQ ID NO: 71), AGY56142 (SEQ ID NO: 72), AGY56143 (SEQ ID NO: 73), AGY56144 (SEQ ID NO: 74), AGY56145 (SEQ ID NO: NO:59), 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

[0332] b.) providing a heterologous S protein or a fragment thereof selected from the genotypes or serotypes of Massachusetts, QX, Q1, Arkansas, variant 2, and Brazil, or selected from the amino acid sequence of SEQ ID NO: 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17, 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:6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17, 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

[0333] c.) replacing the spike protein of 4 / 91 IBV in a) with the heterologous S (spike) protein or a fragment thereof in b) so that 4 / 91 IBV has a heterologous S protein or a fragment thereof; and

[0334] d.) obtaining said 4 / 91 IBV having a heterologous S protein or a fragment thereof; and

[0335] e.) Adding a pharmaceutically acceptable carrier.

[0336] 99. The method of clause 98, wherein the fragment of the heterologous S (spike) protein is the extracellular domain of the spike protein.

[0337] 100. The method of clause 98 or 99, 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.

[0338] 101. The method of clause 98 or 100, wherein the heterologous S protein is a full-length spike protein.

[0339] 102. The method of any one of clauses 98 to 101 wherein 4 / 91 IBV 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 / 4 / 91, UK / 7 / 91, UK / 793B / 91, 4 / 91-pathogenic, 4 / 91 attenuated, IB4-91 and CR88. BRIEF DESCRIPTION OF THE DRAWINGS

[0341] Figure 1 In ovo replication kinetics of CR88 rIBV H52 S 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.

[0342] 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<0.0001) were calculated using GraphPad Prism and a conventional one-way ANOVA test.

[0343] Figure 3 Summary of RT-qPCR results for kidney tissue. Each bird is represented as one data point.

[0344] Figure 4 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.

[0345] Sequence Overview

[0346] SEQ ID NO: 1: CR88 genome

[0347] SEQ ID NO:2: CR88 IBV spike (S) protein.

[0348] SEQ ID NO: 3: CR88 IBV nucleocapsid (N) protein.

[0349] SEQ ID NO: 4: CR88 IBV envelope (E) protein.

[0350] SEQ ID NO: 5: CR88 IBV membrane glycoprotein (M).

[0351] SEQ ID NOs: 6 and 7: Heterologous S protein from the Massachusetts genotype or serotype or fragments thereof.

[0352] SEQ ID NO: 8 and 9: Heterologous S protein from QX genotype or serotype or fragments thereof.

[0353] SEQ ID NO: 10 and 11: Heterologous S protein from Q1 genotype or serotype or fragments thereof.

[0354] SEQ ID NOs: 12 and 13: Heterologous S protein from the Arkansas genotype or serotype or fragments thereof.

[0355] SEQ ID NO: 14 and 15: Heterologous S protein or fragment thereof from variant 2 genotype or serotype.

[0356] SEQ ID NO: 16 and 17: Heterologous S protein from Brazil genotype or serotype or fragments thereof.

[0357] SEQ ID NO: 18: pUC57-s CR88 mIBV.

[0358] SEQ ID NO: 19: IBV H52 spike protein nucleic acid coding sequence.

[0359] SEQ ID NO:20: IBVCR88 spike protein nucleic acid coding sequence.

[0360] SEQ ID NO: 21: pUC57-s CR88 rIBV donor plasmid.

[0361] SEQ ID NO: 22: pUC57-s CR88 rIBV H52 S donor plasmid.

[0362] SEQ ID NO: 23 to SEQ ID NO: 52: Primers.

[0363] SEQ ID NO:53: KF377577 (4 / 91 IBV nucleotide sequence)

[0364] SEQ ID NO:54:AGY56140 (4 / 91 IBV spike protein amino acid sequence)

[0365] SEQ ID NO: 55: EU914938 (4 / 91 IBV spike protein amino acid sequence)

[0366] SEQ ID NO: 56: KM067900 (4 / 91 IBV spike protein amino acid sequence)

[0367] SEQ ID NO: 57: EU780081 (4 / 91 IBV spike protein amino acid sequence)

[0368] SEQ ID NO:58:AGY56143 (4 / 91 IBV E protein amino acid sequence)

[0369] SEQ ID NO:59:AGY56144 (4 / 91 IBV M protein amino acid sequence)

[0370] SEQ ID NO:60:AF093793 (4 / 91 IBV spike protein amino acid sequence)

[0371] SEQ ID NO: 61: Primer Example

[0372] 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.

[0373] Example 1: Generation of recombinant IBV CR88, wherein the coding sequence of the CR88 spike protein is replaced by the coding sequence of a heterologous spike protein or spike protein extracellular domain

[0374] Targeted RNA recombination and rescue of recombinant IBV

[0375] To generate recombinant IBV, the targeted RNA recombination method described by van Beurden et al. (Virol J. 2017; 14(1): 109) was used. A murinized helper virus (mIBV) was generated to allow targeted recombination of IBV in cell culture. Construction of the murinized IBV CR88 donor plasmid

[0376] To generate the CR88 murinized (m)IBV donor plasmid, a donor sequence was synthesized by a commercial supplier: 497 bases from the 5'UTR of the CR88 genome were fused to the 3' portion of the 1ab region (752 bases). The first 72 bases encode the CR88 IBV spike protein, followed by 3753 bases of the extracellular domain of the MHV spike protein, followed by the terminal 210 bases of the CR88 IBV spike protein, and the subsequent sequence continued until the 3' end of the genome. In addition, a SacI restriction site and a T7 promoter sequence were added to the 5' end of the donor region, as well as a 100x polyA sequence, followed by a Not I restriction site at the 3' end for linearization. These were added separately. A silent A to C mutation was introduced at position 5634 of the assembled sequence to generate an XhoI restriction site. The synthesized sequence was inserted into pUC57-simple to generate the pUC57-sCR88 mIBV donor plasmid (SEQ ID NO: 18).

[0377] Rescue of CR88 mIBV

[0378] CR88 mIBV was rescued similarly to H52 mIBV (van Beurden et al. Virol J. 2017; 14(1): 109), with some changes to the protocol: the viral allantoic fluid stock was concentrated by ultracentrifugation, and viral RNA was isolated for electroporation. 18 ml of viral allantoic fluid was passed through a 2 ml 20% sucrose cushion in TNE (Tris, NaCl, EDTA) buffer at 50,000 x g for 2 hours. The supernatant was discarded, and the pellet was resuspended in 150 μl TNE buffer and RNA was isolated using the QIAamp viral RNA mini kit (Qiagen). Furthermore, chicken embryo fibroblasts (CEF) were used for electroporation instead of BHK cells (2 pulses 250 V / 300 μF, 10 seconds apart), and 1.25% DMSO was added to the electroporation mixture.

[0379] Construction of an IBV CR88 donor plasmid in which the coding sequence for the CR88 spike protein is replaced by the coding sequence for a heterologous spike protein or spike protein extracellular domain

[0380] An example of a construction in which the CR88 spike protein is replaced with the H52 spike protein is described. The IBV H52 spike nucleic acid coding sequence (SEQ ID NO: 19) was used as a template to replace the IBV CR88 spike protein nucleic acid coding sequence (SEQ ID NO: 20) in the pUC57-s CR88 rIBV donor plasmid (generated as described above for the mIBV plasmid by keeping the CR88 spike protein sequence (SEQ ID NO: 21) intact). Bases 1657-5151 of SEQ ID NO: 21 were replaced with the corresponding IBV H52 spike protein nucleic acid coding sequence (SEQ ID NO: 19). This created the pUC57-s CR88 rIBV H52 S donor plasmid (SEQ ID NO: 22), in which the IBV H52 spike protein is encoded by bases 1657-5145. To this end, the H52 spike protein nucleic acid coding sequence and the pUC57-s CR88 backbone sequence were amplified in two separate PCR reactions using High-fidelity DNA polymerase (NEB; see Table 1 for primers). The PCR product was purified using the QIAquick gel extraction kit (QIiagen) and then transferred to HiFi DNA Assembly Cloning Kit (NEB) was used for Gibson assembly to generate pUC57-sCR88 rIBV H52 S donor plasmid (SEQ ID NO: 22).

[0381] Table 1: Gibson assembly primers designed by NEB using the NEBuilder online tool and used to generate PCR products to assemble the pUC57-s CR88 rIBV H52 S donor plasmid (SEQ ID NO: 22).

[0382]

[0383] Targeted RNA recombination and rescue of recombinant IBV

[0384] To rescue CR88 rIBV with heterologous spike protein or spike protein ectodomain, LR7 cells were infected with CR88 mIBV and electroporated with in vitro transcripts generated from the pUC57-s CR88 rIBV H52 S donor plasmid after NotI linearization. Subsequently, cells and supernatants were injected into 8-day-old embryonated SPF chicken eggs (VALO BioMedia) and incubated at 37.5°C and 60% humidity for 9 days. Allantoic fluid from eggs with dead embryos was collected and used for endpoint dilution in 8-day-old SPF chicken eggs. MagMAX was used. TM Core Nucleic Acid Purification Kit (ThermoFisher) and KingFisherTM Nucleic acid isolation was performed on limiting dilution allantoic fluid samples using the Duo Prime purification system (ThermoFisher). The nucleic acids were subsequently analyzed for the presence and relative abundance of rIBV by IBV-specific RT-qPCR using the protocol adapted from Callison et al. (J Virol Methods. 2006; 138(1-2): 60-5). Briefly, the same primers and probes were used, and the temperature protocol was adapted to that used. Fast Virus 1-Step Master Mix (ThermoFisher) and StepOnePlus TM Real-time PCR system (ThermoFisher). Allantoic fluid from eggs that tested positive at the highest inoculation dilution was then used to propagate into 8-day-old embryonated SPF chicken eggs. The allantoic fluid was diluted 1:100 in 1xPBS, and 100 μl was injected into each egg. Allantoic fluid was harvested 48 hours after inoculation, cleared of debris, and stored at -80°C. The stock fluid was then analyzed again by RT-qPCR to confirm quality.

[0385] To confirm the correct insertion of the spike protein into the generated rIBV, 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 2 , QIAquick PCR cleanup, and subsequent Sanger sequencing by the supplier.

[0386] Table 2: PCR and sequencing primers to confirm the correct insert and spike protein sequence in CR88 rIBV H52 S

[0387]

[0388] Generation and characterization of CR88 recombinant IBV in which the coding sequence of the CR88 spike protein or spike protein ectodomain is replaced by the coding sequence of the spike protein from another IBV genotype

[0389] The same method as described for the generation of CR88 rIBV H52 S was used to generate and identify CR88 recombinant IBV, wherein the CR88 spike protein coding sequence (bases 1657-5151 in SEQ ID NO:21) or the CR88 spike protein extracellular domain coding sequence (bases 1711-4941 in SEQ ID NO:21) was replaced by the coding sequence of the IBV spike protein or spike protein extracellular domain of the serotype and genotype listed in Table 3.

[0390] Table 3: Primers used for Gibson assembly of H52 rIBV donor plasmids with heterologous spike protein or spike protein ectodomain.

[0391]

[0392]

[0393] If necessary, primers used to identify and differentiate different spike protein sequences in recombinant IBV are adapted to the respective spike protein sequences.

[0394] Example 2: In ovo replication dynamics

[0395] Eight eight-day-old embryonated chicken eggs were inoculated with 102 50% embryo infectious doses (EID 50 ) recombinant IBV and appropriate controls. Eggs were incubated at 37.5°C, 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, 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 HamiltonStarlet 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. Briefly, the same primers and probes were used, and the temperature protocol was adjusted to suit the use Fast Virus 1-StepMaster 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.

[0396] Compared with the recombinant wild-type viruses CR88 and H52, the replication of CR88 rIBV H52 S was confirmed by injecting 100 EID 50were analyzed. Slightly different replication kinetics were observed at early time points. However, after 32 h, all viruses reached comparable ct values. All embryos were alive 32 h after inoculation, whereas at the 48 h post-infection time point, all remaining embryos in the wild-type control group had died. In contrast, 60%-80% of embryos infected with CR88 rIBV H52 S were still alive at the 48 and 72 h post-inoculation time points. The replication of CR88 rIBV H52 S is considered to be equally efficient compared to the wild-type virus, as all viruses reached stationary phase after 32 h (see Figure 1 ).

[0397] Example 3: Preparation of vaccines and challenge viruses

[0398] To demonstrate the efficacy of CR88 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 ).

[0399] Challenge viruses M41, 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 ).

[0400] Example 4: Determination of vaccine efficacy

[0401] 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 (M41, QX, Q1, Ark, variant 2 or Brazil). On the 7th day after the challenge, all chickens were euthanized, pharyngeal swabs were taken, and the 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 4). 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 ciliary movement (scores 3 and 4), the ring was recorded as ciliary arrest positive.

[0402] 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 TMCore 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).

[0403] Table 5: Ciliary arrest scores in tracheal rings

[0404] Cilia activity [%] Ciliary arrest score 100 0 <100-75 1 <75-50 2 <50-25 3 <25-0 4

[0405] Example 5: Efficacy of recombinant IBV 4 / 91 encoding heterologous spike protein or spike protein ectodomain

[0406] The aim of this study was to demonstrate that vaccination with recombinant IBV CR88 (genotype and serotype 4 / 91) encoding a heterologous spike protein or fragment thereof can confer protection against challenge with a spike protein homologous challenge strain.

[0407] The study analyzed whether recombinant IBV CR88 (genotype 4 / 91) encoding the IBV H52 (Mass genotype) spike protein could confer protection against challenge with the virulent M41 strain (genotype Mass), which is considered to be a homologous challenge to the encoded IBV H52 spike protein and a heterologous challenge to the IBV CR88 backbone. All chickens were observed daily for clinical signs. No clinical signs were recorded after vaccination or challenge. Back titration of the 1-day-old inoculation with CR88 rIBV H52 S and H52 rIBV wild-type determined that the titer of the M41 challenge virus applied on day 21 after vaccination was 10 4.38 EID 50 / ml and 10 4.5 EID 50 / ml(target 10 4.3 EID 50 / ml), and 10 4.32 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 5.

[0408] Table 5: 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.

[0409] Group vaccine attack Average ciliary motility arrest score Not affected [%] 1 - - 3,0 100 2 - M41 40,0 0 3 H52 M41 8,9 93 4 CR88 rIBV H52 S M41 8,6 93

[0410] All animals in the strict negative control group showed normal ciliary motility, while all animals in the challenge control group were positive for ciliary arrest. In contrast, 93% of animals vaccinated with either CR88 rIBV H52 S or H52 rIBV wild-type virus were protected. In addition, the viral load in the kidneys of animals vaccinated with CR88 rIBV H52 S was reduced compared with animals in the challenge control group ( Figure 3 ).

[0411] In addition, we analyzed whether recombinant IBV CR88 encoding the IBV QX spike protein could confer protection against challenge with the virulent D388 QX strain, as a homologous challenge agent encoding the IBV QX spike protein and a heterologous challenge agent to the IBV CR88 backbone. All birds were observed daily for clinical signs. No clinical signs were recorded after vaccination or challenge. Back titration of the CR88 rIBV QX S and QX vaccines at 1 day of age determined that the titer of the D388 QX challenge virus exceeded 10,000 at 21 days post-vaccination. 5 EID 50 / ml(target 10 4.3 EID 50 / ml) and 10 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 4 are shown and summarized in Table 6.

[0412] 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 bird 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.

[0413] 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 CR88 rIBV QX S D388 QX 8,9 100

[0414] 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 CR88 rIBV QX S or QX vaccines were protected.

[0415] Similar results were obtained with other CR88 rIBVs with heterologous spike proteins or spike protein extracellular domains.

[0416] This result highlights the suitability of IBV 4 / 91 as an efficient backbone for generating recombinant IBV with heterologous spike proteins, particularly when compared to prior art data for the IBV Beaudette backbone, which showed excellent results. Sequence Listing <110> Boehringer Ingelheim Animal Health GmbH <120> 4 / 91 IBV vaccine with heterologous spike protein <130> 01-3343 <160> 61 <170> PatentIn version 3.5 <210> 1 <211> 27659 <212> DNA <213> Avian infectious bronchitis virus <400> 1 acttaagtgt gatataaata tatatcatac atactagcct tgtgctagat ttccaactta 60 acaaaacgga cttaaatacc tacagttggt ccctataggt gttccattgc agtgcacttt 120 agtgccctgg atggcacctg gccacctgtc aggtttttgt tattaaaata atattgttgc 180 tggtatcact gcttgttttg ccgtgtctca ctttatacat ccgttgcttg ggctacctag 240 tatccagcgt cctactggcg ttgtggtcgg ttcgagtgcg aagaacctct ggttcatcta 300 gcggtacgcg ggtgtgtgga agtagcgctt cagacgtacc ggttctgttg cgtgaaatac 360 ggggtcacct ccccccacat acctctaagg gcttttgagc ctagcgttgg gctacgttct 420 cgcacaaggt cggctatacg gcgtttgtag ggggtagtgc caaacaaccc ctgaggtgac 480 aggttctggt ggtgtttagt gagcagacat acaatagaca gtgacaacat ggcttcaagc 540 ctaaaacagg gagtatctcc caaaccaagg gatgtcattc ttgttgccaa agacattccc 600 gaacaactcc gtgacgcttt gtttttctat acgtcacata 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tgacattcct 1500 gttgagaaag aaggttggtc tgcaattttt gaaggacatc tttgctatgt ctttaagagc 1560 ggtgaccgtt tttatgcggc acctctttct ggaaattttg cattgcatga tgtacattgt 1620 tgtgagcgtg ttgtctgtct gtctgatggt gtaacaccgg agataaatga tggactcatt 1680 ctagcagcaa tttattcatc tgttagtgtt tcagaactcg tggcagcact taaaaagggc 1740 gaaccattta agttcttggg gcataaattt gtgtacgcga aggacgcagc agtgtcgttt actttagcga aggctgccac tattgcagat gtactgaagc tgtttcaatc agctcgtgtg caagcggag atgtgtggtc tgcatttact gaaaagtctt ttaatttctg gaaactcgca tatggaaag tgcgtaatct tgaagaagtt gtgaagactc atttttgtaa agctcaaatg tcacttatcg ttctagtagc agtgcttgga gaaggtgttt ggcatattgc ttctcaggtc atctacaaat tagtggtct ttttactaa gttgttgact tttgtgaaaa acactggaaa ggttttgtg tacagttgaa gaaggctaag ctcattgtga ctgaaaatct ttgtgttctt aagggagtgg cacagcattg ttttcaacta ttgctggatg caatacattc tttgtatatg agttttaaga agtgtgcact tggtagaatt catggagact tactcttctg gaaagggggt gtacacaaaa ttgttcaaga tggcgatgaa gtttggtttg acgccattga tagtattgat gttgaagatc tgggtgttgt caaagaaaaa cctatagatt ttgaggtttg tgatgacgta acacttccag aaaatcaacc cggtcatatg gttcaaatcg aggatgacgg aaagaactat atgttcttcc gcttcaaaaa ggatgagaat atttattata caccaatgtc tcaacttggt 2520 gcaattaatg tagtttgcaa agcaggcggc aaaaccgtta cctttggaga cattacagtg 2580 aaagaaatac cgccacctga tgtcgtgcct attaaggtta gcatagagtg ttgtggtgag 2640 ccatggaata caatcttcaa gaaagcttat aaagagccca ttgaagttga gacagacctc 2700 acagttgaac aattgctctc tgtgatttat gagaaaatgt gtgacgacct caaactgttt 2760 ccagaggcac ctgagcctcc accatttgag aatgttgccc ttgttgataa gaacggtaaa 2820 gatttggatt gcataaaatc atgccacctt atctaccgtg attgtgagag cgatgatgac 2880 atcgaggaag aagatgctga agagtgtgac accgattcag ctgatgctga agcatgtgac 2940 acagcctcag agtgtgaaga agaggacgag gatactaaag tgttggctct tatacaagac 3000 ccagcaagta ataataccc tcttccactt gatgatgatt atagcgtcta taatggatgt 3060 attgttcata gagacgctct tgatgtagtg aatctaccat ctggcgaaga aacttttgtt 3120 gttaataact gttttgaggg agctattaaa ccactcccac agaaagttat tgatgttcta 3180 ggtgactggg gcgaggctgt tgatgcacaa gaacaattgt gtcaacagga atctacccaa 3240 gtcacacttg agaaaccagt tgagggtcct actggtattt gtcacgtaat ggctgaacaa 3300 gctgttgttg atgaacagga ggtagtttct gtatttgaag aaaagtctga ggttattgtt 3360 tacacacctg cagatctgga agttgttaaa gaaacagcag gagaacttga tgagttcatt 3420 ctcatctttg atgtccctaa agaagaagtt gtgtctcaag agaaagataa gccacaggtt 3480 gagcaagagc ctactcaagt tgttaaacca caacgtgaga agaaggctaa aaagttcaga 3540 gttaaaccat ctacatgtga aaagcccaaa tttttggagt acaaaacatg tgtgggtgat 3600 ctgactgttg taattgctaa ggcattggat gagtttaaag agttctgcat tgtaaacgct 3660 gcaaatgaac atatgtctca tggtggtggc gttgcgaaag caattgcaga cttctgtgga 3720 cctgattttg tggaatattg tgaggcctat gttaagaaac atgggccaca acagagactt 3780 gttacacctt cgtttgtcaa gggcattcaa tgtgtgaaca atgttgtggg acctcgtcat 3840 ggagacagca acttgcgtga gaaactcgtt gttgcttaca agaatgttct tgtggatggc 3900 gtcttcaact atgtagtgcc agttctctct tcagggattt ttggtgtgga ttttaaaatg 3960 tcaatagacg caatgcgtga agcctttgat ggttgcgaca tacgcatcct tttgttttct 4020 ctgagtcaag aacacatcga ctattttgat gtaacttgca agcagaagac aatttatctt 4080 acggaggatg gtgttaaata ccgctccgtt gttgtaaaac ctggcgactc attgagtcaa 4140 tttggacagg tttttgctaa aaataaggca gtctttacag ctgatgatgt tgaggataaa 4200 gaaatcctct tcgttcctac tactgacaag actgttcttg attattatca tttagatgcg 4260 caaaagtatg tgatatattt acaagctctt gcgcagaaat gggatgttca atatagggac 4320 aattttattg cattagagtg gcgtgatgga aattgctggg ttagttcagc aatagtgctc 4380 cttcaggctg ctaaggtaaa gtttagaggt tttcttgcag aagcatgggc taagtttttg 4440 ggtggtgatc ccactgattt tgtggcctgg tgttatgcaa gttgcaatgc taaagttggt 4500 gagttttcag atgctaattg gctcttggct aatctggcag aacattttga tgcagattac 4560 acgaatgcac ttcttaagaa gcgcgtatca tgtaactgcg gtgttaagag ttatgagctt 4620 aggggtcttg aagcttgcat tcaaccagta agggcaccta accttctaca ttttaagaca 4680 caatattcaa attgcccaac ctgtggcgca aatagtacgg atgaagtaat agaagcttca 4740 ttaccttact tattgctctt tgctaccgat ggtcctgcta cagttgattg tgatgaaaat 4800 gctgtaggga ctgttgtttt cattgggtct actaatagtg gacactgtta tacacaagcc 4860 atgggtaagg cttttgataa tcttgctaaa gatagaaaat ttggaaagaa gtcgccttac 4920 attacagcaa tgtacacacg cttctccctt aagagtgaaa accctttgcc tgtcaagcag 4980 agccagggta aaactaaagt agtaaaagaa gatgtttcta atctagcaac aagttcaaaa 5040 gtcagttttg atgatcttac tgactttgag cagtggtatg atagcaacat ctatgaaagt 5100 cttaaagtgc aggaaacgcc tgataattct gatgggtatg tgtcatttac aacaaaagaa 5160 gattctaagt tgccactgac acttaaagtt agaggtatta aatcagttgt tgattttagg 5220 tcgaaggatg gtttcactta caaattaata cctgatattg atgaaaattc aaaagcacca 5280 atttactatc cagtcttgga ctctattagt cttaaggcaa tatgggtgga aggtggtgct 5340 aattttgttg ttgggcatcc aaattattat agtaaatctc tccgcattcc tactttttgg 5400 gaaagtgcag agagttttgt taaaatgggt gataaagttg atggtgtaac tatgggcctt 5460 tggcgtgcag agcatcttaa caaacctaat cttgagaaaa ttttcaacat tgctaagaaa 5520 gctattgttg gatctagtgt tgttactacg caatgtggta aagtaattag taaggcggct 5580 acattcattg ctgataaaat aggtggggt gtagttcgca acatattaga tagaattaag 5640 ggtctttgtg ggtttacacg tgggcatttt gaaagaaaat tgtctccaca attcataaaa 5700 acacttatat tcttcttctt ctactttata aaggctagtg ctaagggttt agttgctagc 5760 tataagaatg tgttatgtaa ggtggtattc gctactttat ttatagtgtg gtttatatat 5820 acaagtaatc footgtgtt tactggaaca cgtatgttag acttcctatt tgagggttct 5880 ttgtgtggtc cttataatga ctacggtaaa gattcttttg atgtgttacg ctattgtgga 5940 gatgatttta cttgtcgtgt gtgtttacat gacagagact ctctacattt gtataaacat 6000 gcttatagca tagaacagat ttaataaagct gcaagttctg gcattgtttt taattggaat 6060 tggctttatt tggtctttct aattttattt gttaagccag tggcaggttt tgttattatt 6120 tgctattgtg tgttttt agtgttgagt tcaactgtgc tccaaactgg tgtaggtttt 6180 ctagactggt ttattcaaac agtttttact cactttaatt ttgggtgc aggttctat ttctggctct tttataaatt gtacatacag gttcatcata tactgtattg tagtata acatgtgaag tgtgtaagag agttgcacgc agtaacaggc atggggttag tgttgttgtt ggtggacgca agcaaattgt gcatgtgtac actaactctg gttacaactt ttgtaagaga cataattggt attgtaggaa ttgtgatgta tatggtcacc aaaacacatt tatgtctcct gaagttgctg gtgagctgtc tgaaaagctt aaacgccatg ttaaacctac agcacatgct taccacgttg tggatgaggc ttgcgtagtt gatgattttg ttaacttaaa atacaaagct gcaactcctg gtaaggatgg tgcacctcct gcagttaaat gtttcagtgt tacagatttc ttgaagaaag ctgtttttct taggcg ctgaaatgtg aacaaatatc tagtgtgt tttatagtgt gtaatacgca gagtgcgcat gctttagagg aagcaaagaa tgcagccatc tattatgcgc aatacctgtg taaacctata cttatactcg accaggcact ctaccagaat 6840 ttaatagtgg aacctgtatc gaagagcgtt gtcaacaaag tgtgtgacat tttgtctagg 6900 ataatttctg tagatactgc atctttggat tataaagcag gtacagttcg tgatgccttg 6960 ctgtctgtta ctaaagatga agaagctgta gatatggcta tcttctgtca taatcatgaa 7020 gttgaatata caggtgatgg ttttactaat gttataccgt catatggtat agaccctgat 7080 aaattaacac ctcgtgatag agggttttg ataaatgcag atgcttctgt tgctaactta 7140 agagttaaaa atgctccgcc ggtagtatgg aagttttctg atcttattaa gttgtctgac 7200 agttgtctta aatatttaat ctcagcaact gtcaagtcag ggtctcgttt ctttataaca 7260 agatctggtg ctaaacaaat ttttcctgt agtactcaga aattgttggt agagaaaaag 7320 gctggtggtg tcattagtgg tacctttaat tggtttaaga gttgttgtaa atggctcttg 7380 atcttctatg tgctttttac attgtgttgt ttgggttgtt atcatatgga gacgaataaa 7440 agttttgttc atcctatgta tgatgttaac tctacaatgc atgttgaagg ctttaaggtt 7500 atagataaag gtgttattag agacattgta ccagaggatg cttgtttctc tataagtttt 7560 gctaactttg atgcatttg gggtaaacca tatgtgaata gtagagactg tccaattgtt 7620 acagcagtca tagatggcgc tggaacaata gcagctggtg ttcctggttt tgtagactgg 7680 gttcttgatg gtgttatgtt tgtacacatg acacaaacag aaagaaaacc ctggtacatt 7740 cccacgtggt ttaacagaga aattgttggt tacactcagg attcaattat tactgaaggt 7800 agtttttata catctatagc gttgttttct gctaggtgtt tatacttaac agccagtaat 7860 acaccgcaat tgtattgttt taatggtgat aatgatgctc ctggagcctt accatttggt 7920 agtattgttc ctcataaggt ctacttccaa ccaaatggtg ttaggcttgt agttccacaa 7980 caaatactac acacacccta catagtgaag tttgtttcag acagctattg tagaggtagt 8040 gtatgtgagt atactaaacc aggttactgt gtgtcattga actcacagtg ggttttgttt 8100 aatgatgaat acacaagcaa acctggcgttt ttctgtggtt ctactgttag agaacttatg 8160 ttaataatgg ttaatacatt cttactggt gtcaatccta atatttatat gcaactagca 8220 actatgtttt tgatactagt tgttgttgtt ttaatttttg caatggttat aaagtttcaa 8280 ggtgttttta aagcttatgc gaccattgtt tttacaataa tgttagtttg ggttgttaat 8340 gcatttgttt tgtgtgtaca tagttataac agtgttttag ctgttatatt attggtaatc 8400 tattgttatg catcattggt tacaagtcgt aatactgcta tataatgca ttgttggctt 8460 gtgttacct ttggtttaat tgtacccata tggttggcgt gttgctacct ggcatttgtt 8520 ttatatatgt acacaccatt gttttctgg tgttacggta ctactaaaaa tactcgtaaa 8580 ttgtatgatg gcaacgagtt tgttggtagt tatgatcttg ctgcgaagag cactttttgtt 8640 attcgcggtc ctgaatttgt taagcttacg aacgagatag gtgataagtt tgaacactat 8700 ctctcagcgt atgctagact taatactac tcaggcactg gcagtgaaca agattacttg 8760 caagcctgtc gtgcatggtt agcttatgct ttggaccagt atagaatag tggtgtggaa 8820 attgtgtata ctccaccacg ttactctatt ggtgttagta gattacaggc tggttttaag 8880 aaactagttt ctcctagtag tgctgttgaa aggtgcattg ttaggtctc ttatagaggt 8940 aataagctta atggactgtg gctaggtgat actatctact gtccgcgaca tgttctaggc 9000 aagttttcag gcgatcaatg gagtgatgta cttaatcttg ctaataatca tgagtttgag 9060 gttgtaactc aaaatggtgt tactttgaat gttgttagta gacggttgag aggcgcagtt 9120 ttaatttac aaactgctgt cgccaatgct gacactccta agtataagtt tgttaaagct 9180 aattgtggag atagtttcac aattgcttgt tcttatggtg gtacagttgt gggactctac 9240 cctgttacta tgcgttctaa tggtactatt agagcgtctt tccttgcagg agcttgtggc 9300 tcagttggtt ttaatataga aaagggtgta gttaatttct tttatatgca ccatcttgag 9360 ttacctaatg cattacacac cggaactgac ctagcgggtg acttctatgg tggttatgtg 9420 gatgaagagg ttgcacaaag ggtgccacca gataatttag ttactaacaa tattgtagca 9480 tggcttatg cagcaattat tagtgttaaa gagagtagct tctcactgcc taaatggttg 9540 gagagtacta ctgccagtgt tgaagattat aataagtggg ctggtgataa tggttttaca 9600 ccattttcta ctagtactgc tattactaaa ttaagtgcta taacgggagt tgatgtttgt 9660 aaactccttc gcactattat ggtaaaaagt agtcaatggg gtagtgatcc cattttagga 9720 caatacaatt ttgaagatga attgacacct gagtctgtct ttaatcaggt tggcggtgtt 9780 agattacagt cttctattgt aagaagagcc acatcttggt tttggagtag atgtgtgtta 9840 gcttgcttct tgtttgtgtt gtgtgctgta gttttgtta cggcagtacc acttagatat 9900 tatttacatg cagctgttat tttgttagtg gctgtacttt ttatttcttt tactgttaaa 9960 catgtatgg catatatgga tacttttcta ttgccgacat tggttacagt tattattgga 10020 gtttgtgctg aagttccttt catatacaat actctaatta gtcaagttat tattttctta 10080 agtcaatggt atgatcctgt agtttttgat actatagtac catggatgtt gttgccatta 10140 gtgttgtaca ctgcttttaa gtgtgtacaa ggttgctata tgaattcgtt caatacttcg 10200 ttgttaatgc tgtaccagtt tatgaagtta ggttttgtta tttatacctc ttctaacact 10260 cttactgcat atacagaagg taattgggag ttattctttg agttggttca cactactgtg 10320 ttggctaatg ttagtagtaa ttccttaatt ggtttaattg tctttaagtg tgctaagtgg 10380 atgttgtatt attgtaatgc aacatacttt aacaattg ttttaatggc agttatggtt 10440 aatggcatag gctggctttg cactgttac ttggattgt attggtggat taataggtt 10500 tttggtttaa ccttaggtaa atacatttt aaagtttcag tagaccata taggtatatg 10560 tgtctgcata agatagaccc ccctaaaact gtgtgggaag tctttcgac aaatactt 10620 atacaaggga ttggtggtga ccgtgtgttg cctattgcta cagttcaatc taaattgagt 10680 catgtaagt gtacaactgt tgtgttaatg caactttga ctaagcttta tgttgagca 10740 aattcaaaaa tgcatgctta tctgttgag ttacacaata aaatcctcgc atctgatgat 10800 gttggagt gcatggataa tttattgggt atgcttatta cactattttg tatagattct 10860 actattgatt taggtgagta tgtgatgat attacktaga ggtcaactgt attacaatca 10920 gttactcaag agttttcaca cataccctct tatgctgaat atgaagagc windowatctt 10980 tatgaaaagg tttagctga ttctaaaaat gtggtgtaa cacagcaaga gcttgctgca 11040 tatcgtaaag ctgccaatat tgcaagtca gttttgata gagatttggc tgttcaaag 11100 aagttagaca gcatggcaga acgtgctatg acaacaatgt ataaagaggc gcgtgtaact 11160 gatagaggag caaaattagt ctcatcacta catgcgttac ttttttcaat gcttaagaaa 11220 atagattctg aaaagcttaa tgtcttattt gatcaggcta gtagtggtgt tgtacctcta 11280 gctactgttc caattgtttg tagtaataag cttacccttg tagtaccaga tccagaaact 11340 tgggtcaagt gtgtggaagg tatgcatgtt acatattcaa cagttgtttg gaatatagat 11400 actgttattg atgctgatgg tacagaggta catccaactt ctacaggtag tggattgaca 11460 tactgtataa gtggtgccaa tatagcatgg cctttaaagg ttaacttgac taggaatggg 11520 cataataagg ttgatgttgc tttgcagaat aatgagctta tgcctcatgg tgtaaaaaca 11580 aaggcttgcg tagcaggtgt ggatcaagca cattgtagcg tagagtctaa atgttattat 11640 acaaatatta gtggcaattc agttgtagct gccattactt cttcaaatcc aaatctgaaa 11700 gtagcttcat ttttgaacga ggcaggcaat cagatttatg tagacttaga cccaccatgc 11760 aaatttggca tgaaggtggg tgacaaggtt gaggttgttt acttgtattt tataaagaat 11820 acaaggtcga ttgttagggg tatggtactt ggtgctatat ctaatgttgt tgtcttacag 11880 tctaaagggc atgaaacaga ggaagtggat gctgttggca ttctttcact ttgttcttttt 11940 gcagtagatc ctgcagacac gtattgtaaa tatgtggcag caggtaatca acctctaggt 12000 aactgtgtta aaatgttgac agttcataat ggtagtggct ttgctataac atcgaagcca 12060 agtccaactc ctgatcagga ttcctacgga ggagcttctg tgtgtctcta ttgtagggca 12120 cacatagcac atcctggtgg tgcaggaaat ttagatgggc gttgtcaatt taaaggttcc 12180 tttgtgcaaa taccaactat ggagaaagac cctgttggat tctgtctacg tataaggtt 12240 tgcactgtct gtcaatgttg gattggttat ggatgtcagt gcgatgcact tagacaacca 12300 aaaccttctg ttcaatcagc tgccaatgta gctgatttcg ataagaatta tttaaacggg 12360 tacggggtag cagtgaggct cggctgatac cccttgctag tggatgtgat cctgatgttg 12420 tgaagcgagc ctttgatgtt tgtaataagg aatcagccgg tatgtttcgt aatttgaagc 12480 gtaactgtgc gcggttccaa gagtacgtg atactgaaga tggaagtctt gagtattgtg attcattctt cgtggttaaa caaaccactc ctagtaatta tgaacatgag aaagcttgtt atgaagactt aaagtcagaa gtgacggctg accatgattt ctttgtgttc aatagaaca tttataatat tagtagcaa cgtcttacta agtatactat gatggatttt tgctatgctt tgaggcattt tgacccaaag gactgcgaag ttcttaaaga aatacttgtc acttatggtt gtatagaaga ttatcaccct aagtggtttg aagagaataa ggattggtac gacccaatag aaaacccaaa atattatgcc atgttggcta aaatggggcc tattgtacga cgtgctttat tgaatgctat tgagttcgga aaccttatgg ttgagaaagg ttatgttggt gtagttacac ttgacaacca agatcttaat ggcaaatttt atgactttgg tgattttcag aaaacagcac ttggtgctgg ggttcctatt tttgatacat attattccta catgatgccc atcatagcca tgacggatgc tttagcacct gaaaggtatt ttgaatga tgtgcataag ggttataagt cttatgatct tctcaagtat gattatactg aggagaaaca agatttgttt cagaagtact 13200 ttaagtattg ggaccaggag taccatccta actgccgtga ctgtagtgat gacaggtgtt 13260 tgatacattg tgcaaacttc aacatattgt tttctacatt gataccacag acttcttttg 13320 gtaatttgtg tagaaaagtg tttgttgatg gtgtaccttt tatagctact tgtggctatc 13380 attctaaaga acttggtgtt attatgaatc aagataacac catgtcgttt tcaaaaatgg 13440 gtttaagtca actcatgcag tttgttggag accctgcctt gttagtggga acatccaata 13500 atttagtcga tcttagaacg tcttgtttta gtgtttgtgc attagcgtct ggtattactc 13560 atcaaacggt aaaaccgggt cactttaaca aggatttcta tgattttgca gagaaggccg 13620 gtatgtttaa agagggttct tctataccac ttaaacattt cttctaccca caaactggta 13680 atgctgctat aaacgattat gattactatc gttataacag gcctaccatg tttgatatac 13740 gtcaacttct attttgttta gaagtgactt ctaaatactt tgactgttat gaaggcggct 13800 gtataccagc aagccaagtt gtagttaata atttagataa gagtgcaggc tacccattta 13860 ataaatttgg aaaagctcgc ctctattatg aaatgagtct agatgaacag gaccaactct 13920 ttgagagtac aaagaagaat gtcctgccta ctataactca aatgaattta aaatatgcca 13980 tatccgcgaa aaatagagcg cgtacagtgg caggtgtgtc tatcctttct actatgacta 14040 ataggcaatt tcatcagaag attcttaagt ctatagttaa cactagaaat gctcctgtag 14100 ttattggaac aaccaagttt tatggcggtt gggacaatat gttgaggaat ctgattcaag 14160 gtgttgaaga ccctattctt atgggttggg attatcctaa gtgtgataga gcaatgccaa 14220 atttgctgcg tatagcagca tccttggtac ttgctcgcaa acacactate tgttgtactt 14280 ggtctgaacg catttatagg ttgtataatg aatgcgctca ggttttatct gaaactgttt 14340 tagctacagg tggtgtttat gtaaaacctg gtggtactag cagtggtgat gctactactg 14400 cttatgcaaa cagcgttttc aacataatac aagccacatc tgctaatgtt gcgcgtcttt 14460 tgagtgttat aacgcgtgat attgtttatg atgacattaa gagcctgcag tatgagttgt 14520 accagcaggt ttataggcga gttaattttg acccagcctt tgtagaaaag ttttattctt 14580 acttatgtaa gaatttctct ttgatgatct tgtctgacga tggtgttgtt tgttacaaca 14640 acacattggc caaacagggt cttgttgcgg acatttctgg ctttagagaa attctctact 14700 accaaaataa tgtttattg gctgattcta agtgttgggt tgaaccagac ttagaaaaag 14760 gcccacatga attttgttca cagcacacaa tgctagtgga ggtggatggt gagcctaagt 14820 atttgccata tccagatccc tcacgcattt tgggtgcatg tgtctttgta gatgaagtgg 14880 ataagacaga acctgtggct gttatggagc gttatatagc tcttgccata gacgcttacc 14940 cactagtaca tcacgaaaat gaggagtaca ggaaggttt ctttgtgctt ctttcatata 15000 tcagaaaact ctatcaagag ctttctcaga atatgcttat ggactactct tttgtgatgg 15060 atatagatacaa gggtagtaaa ttttgggaac aggagttcta tgagaatatg tatagagccc 15120 ctacaacttt acagtcttgt ggtgtttgtg tagtgtgcaa tagtcaaact atactacgct 15180 gtggtaattg tattcgaaaa ccatttttgt gttgtaaatg ttgttatgac catgtcatgc 15240 acacagacca taaaaatgtt ttgtctataa acccatatacat ctgttcgcaa cccggttgcg 15300 gtgaagcaga tgttactaaa ttataccttg gaggtatgtc atacttctgt ggtaatcata 15360 aaccaaaatt gtcaataccg ttagtatcta atggtactgt ctttggtatt tacagggcta 15420 attgtgctgg tagtgaaaat gttgatgatt ttaatcaact agctactact aattgggcta 15480 ctgtagaacc ttatatttta gcaaatcgtt gtagtgactc attgagacgc tttgctgctg 15540 aaacagtaaa agccacagag gaattacata aacagcaatt tgctagtgct gaggtgagag 15600 aagtactctc agatcgtgaa ttgatcctat catgggagcc aggtaaaacc aggccaccat 15660 tgaatagaaa ttatgttttc acaggctatc actttacaag aactagtaag gtgcagcttg 15720 gtgattttac ctttgaaaaa ggtgaaggta aagatgttgt ctattataga gcaacgtcta 15780 ctgctaaatt gtctgttgga gacatttttg ttttaacctc acacaatgtt gtttctcttg 15840 tagcgccaac gttgtgtccg cagcaaacct tttctaggtt tgtgaattta agacctaatg 15900 taatggtacc ggaatgtttt gtaataataaca ttccacttta ccatttagta ggtaagcaga 15960 agcgtactac agtacaaggt cctcctggca gtggtaagtc acactttgct ataggccttg 16020 ctgcttactt tagtaacgcg cgtgttgtct ttactgcttg ttctcatgca gctgttgatg 16080 ctttatgtga aaaagctttt aagtttctta aagtcgatga ttgcactcgg atagtacctc 16140 aaaggactac tgtcgagtgc ttttctaagt ttaaagctaa cgacacaggc aaaaagtaca 16200 tttttagtac tataaatgcc ttgccagaag ttagttgtga cattcttttg gttgatgagg 16260 ttagtatgtt gaccaattac gaattgtctt ttattaatgg taagataaat taccaatatg 16320 ttgtgtatgt aggtgaccca gctcaattac cggcacctcg taccttactt aatggctcac 16380 tttcaccaaa ggattataat gttgtcacaa accttatggt ttgtgttaaa cctgatatat 16440 tccttgcgaa gtgttaccgt tgtcctaagg aaattgtaga cactgtgtct actctagtat 16500 atgatggaaa gtttattgca aataacccag aatcacgtca gtgtttcaag gttatagtta 16560 ataatggtaa ttctgatgta ggacatgaaa gtggttcagc ctacaacaca actcaattag 16620 aatttgtgaa agattttgtt tgtcgcaata aggaatggcg ggaagcaaca ttcatttcac cttacaatgc tatgaaccag agagcctacc gtatgcttgg acttaatgtt cagacagtag actcatctca aggttcggag tatgattatg ttattttctg tgtaactgca gattcgcagc atgcactgaa tattaacaga tttaatgtgg cgcttacaag agctaagcgt ggtatactag ttgtcatgcg tcagcgtgat gaattgtact ctgctcttaa gttcagag ctaaatagtg aagcaagtct gcaaggtaca ggtttgttta aaatttgcaa caaagaattt agtggtgttc atcctgctta tgcagtcaca actaaggctc ttgctgcaac ttacaaagtt aatgatgaac ttgctgcact tgttaatgtg gaagccggtt cagaatac father cttatttctc ttctaggatt tagtgagt gttaatgttg aaggttgcca taacatgttt ataacacgtg acgaggcaat tcgcaacgta aggggtggg taggattcga tgtagaagcc acacatgctt 17280. gtggtactaa cattggcact aacttacctt ttcaagtagg tttctcgact ggtgcagact ttgtagttac acctgaggga cttgtagata cttcgatagg caataattttt gagcctgtga 17340 attccaaagc acctccaggt gagcaattta atcacttgag agctttattt aagagtgcta 17400 aaccttggca cgttataaga ccaaggatag tgcaaatgtt agcagataat ctatgcaatg 17460 tctctgattg tgtagtgttt gttacttggt gtcatggcct agaactaact acgttgcgct 17520 atttgttaa aataggcaaa gaacaattgt gttcgtgtgg ttctagagct acgactttta 17580 attcacatac tcaagcttat gcttgttgga ggcattgttt gggttttgat tttgtctata 17640 acccacttt agtggatatt caacaatggg gttactcagg taatctacaa tttaaccatg 17700 gtttgcattg taatgtgcat ggacatgccc atgtggcttc tgcggacgcc attatgacgc 17760 gttgccttgc aattaacaat gcattttgtc aagatgtcaa ctgggatttg acatatcctc 17820 atattgcaaa tgaggatgaa gtcaactcta gttgcggta tttacagcgc atgtatctta 17880 atgcatgtgt tgatgctctt aaagttaatg ttgtctatga tataggcaac cctaaaggta 17940 taaagtgtgt tagacgtgtg gatgtcaatt ttagattcta tgataagaat ccaattgtac 18000 18060. ccaacgtcaa acagtttgag tatgactata atcagcacaa ggataagttt gctgatggtc tttgtatgtt ttggaattgc aatgtggatt gttatcctga caattcacta gtctgtagat acgacacacg aaatttgagt gtgtttaatc taccaggttg taatggtggt agtctgtatg tgaacaaaca tgcatttcac acacctaaat ttgaccgcat tagctttcgc aatttgaaag ctatgccatt tttcttctat gattcatcgc cttgtgaaac cattcaagtt gatggagttt cacaagacct tgtgtcatta gctactaagg attgtatcac aaaatgcaac attgggggtg ctgtttgtaa aaagcacgct cagatgtatg cagagtttgt gacttcttat aatgcagctg ttacagctgg ttttactttt tgggttacta father cccttataat ttgtggaaaa gtttttcagc tctccagtct attgacaata ttgcttata tatgtataag ggtggtcatt atgatgctat cgcaggagaa atgcccacag tcataactgg agataaagtt tttgttatag atcaaggtgt agaaaaggca gtttttgtta atcaaacaac actgcctacg tctgtggcgt ttgaattgta tgcgaagaga aatattcgca cactgccaaa caaccgtatt ttgaagggtc 18720 ttggtgtaga tgtaaccaat ggttttgtaa tttgggatta tgctaaccag acaccactat 18780 atcgtaatac tgtcaaggta tgtgcataca cagacattga gccaaatggc ctaatagttc 18840 tgtatgatga tagatatggt gattaccagt cttttcttgc tgctgacaat gctgttttaa 18900 tttctacaca gtgtataag cggtattcat atgtagaaat accatcagat ttgcttgttc 18960 agaatggtat gccactaaaa gatggagcga acctatatgt ttataagcgc gttaatggtg 19020 tgtttgttac actacccaac actttaaaca cacagggccg caattatgaa acttttgaac 19080 ctcgtagtga cgttgagcgt gattttctcg acatgtcaga ggaagatttt gtagaaaagt 19140 atggtaaaga cttaggtcta caacacatac tgtatggtga agttggtaaa ccacaattgg 19200 gtggtttaca cactgttata ggtatgtaca gacttttacg tgcgaataag ttgaatgcaa 19260 agtctgtcac taattcggat tctgatgtca tgcaaaatta ttttgtgttg gcagacaatg 19320 gttcttacaa gcaagtgtgc actgttgtgg atttactgct tgatgattc ttagaacttc 19380 ttaggaacat actgaatgag tatggtacta ataagtcaaa agttgtaaca gtgtcaattg 19440 attaccatag cataaatttt atgacttggt ttgaagatgg cagtattaaa acatgttatc 19500 cacagcttca atcagcgtgg acatgtggtt ataatatgcc tgaactttat aaagtccaga 19560 attgcgttat ggaaccttgc aacattccta actatggtgt tggaataacg ttgccaagtg 19620 gtattatgat gaatgtagca aagtatacac aactttgtca atatctttcg aaaacaacaa 19680 tgtgtgtgcc gcataatatg cgagtaatgc atttggagc aggaagtgac aaaggagtgg 19740 ccccaggtag cgctgttctt aggcagtggc ttcccgaagg tacactcctt gtcgataatg 19800 atattgtaga ttatgtatct gatgcacatg tctctgtgct ttcagattgc aataaatgta 19860 aaacagagca caagtttgat cttgtgatat ctgatatgta tacagataat gattcaaaga 19920 gaaagcatga aggcgttgta gccaataacg gcaatgatga cgtcttcata tacctttcaa 19980 actttcttcg taacaattta gctctgggag gcagttttgc cgtaaaagta acagagacaa 20040 gttggcatga gagtttatat gacattgcac aggattgtgc atggtggaca atgttttgta 20100 cagcagtgaa tgcttcttcg tcagaagcat tcttgattgg tgttaattat ttgggtgcaa 20160 gtgaaaaggt tagagttagt ggtaaaaccc tgcacgcaaa ttatatattt tggaggaatt 20220 gtaattattt acaaacttca gcctatagta tatttgacgt tgctaagttt gatttgaaat 20280 taaaagcaac gccagttgtg aatttgaaaa ctgaacaaaa gacagactta gtctttaatt 20340 taattaagtg tggtaagtta ctggtaagag atgttggaca aaccgctttt actagtgact 20400 ctttggtatg cactatgtag tgctttgctc tatgataata atacttacgt ttactactac 20460 caaagtgcct ttaggcctgg tccaggttgg cacctatatg ggggtgctta tgcagtagat 20520 agggttttta atgaaaccaa caatgcaggc agtgcatctg attgcactgc tggtacttttt 20580 tatgaaagcc ataatatttc tgcttcttct gtagccatga cagtaccaca taatggtatg 20640 tcttggtcag cttcacaatt ttgtacagct cattgtaact tctcagactt tacagtgttc 20700 gttacgcatt gttttaaaaa tcaactcggt agttgtccct tgacaggtat gattcctcag 20760 aatcatattc gtatttctgc tatgagagat ggagttttgt ttataactt aacagttagc 20820 gtatctaaat accctagatt taaatcgctt caatgtgtta gcaattctac atctgtctat 20880 gtaaatggtg accttgtttt cactctaat gaacttctt acgttacggg tgcaggcgtt 20940 tattttaaaa gtggtgggcc tgtaacttat aaagttatga aagagttaa agccctagcc 21000 tactttatta atggtaccgc acagaggtt atttttgtg atactcacc taggtttg 21060 cttgcatgtc agtatacac tggtatttt tcagatggat tctacccttt tactaatcat 21120 tctttagtta aggataggtt tattgtatat cgagaagta gcactacac tactttaaag 21180 ttaactatt tcagtttac taatgtaagt atgctctc ctaattcagg tggcgttgat 21240 actttccaat tatatcaac aagtactgct caggatggtt attataattt taatttatca 21300 tttctgagta gttttgtgta taaaccatct gattttgt atgggtcata caccacat 21360 tgtaagttta gaccagagaa tattatat ggcttatggt ttaattcatt atctgtgtca 21420 cttacttacg gacccattca aggtggttgt aagcaatctg tttttagtaa tagagcaact tgttgctatg cttattctta tcaagggcct agtaggtta agggtgttta tagggggag 21540. ctaacgcaat actttgaatg tggacttcta gtttacgtaa ctaagagtga tggctctcgt attack attack actggtgtta act attack attack cattacttta fathergtgtg ttgagtata fatheragggttggtc aaggttttat tactaatgta actgaagcaa ctgctaatta tagttatcta gcagatggtg gtttagctat tttagatacc 21840. tcaggagcca tagacatatt tgttgttcaa ggtgcatatg gtcttaatta ttataaggtt aatccctgtg aagatgttaa ccaacagttt gtagtgtctg gtggcaactt agttggcatt cttacatctc father aggttctga tctattga accagtttta catcaaactc actaacgga cacgtcgctc tagacgttct gttactggga atgttacaaa ttgcccttat gttagttatg gcaagttttg fatheraacca gatggttctt tatctaat father gaattagaac agtttgtggc gcctttattc aatgttactg agcatgtgct catacctgat agttttaatt taactgtcac agatgagtac atacaaactc gtatggataa ggttcaaatt 22200 atttgccttc agtatgtttg tggtaattct attgaatgca gaaagttgtt tcagcagtat 22260 ggacctgttt gtgaataat attgtctgtt gtaaatggtg taggtcaaag agaggatatg 22320 gaacttttaa gtttctattc ttctactaaa cctagtggtt acaatacacc aatttttaat 22380 aatgtagca ctggtgactt tatattcg ctcctactaa caccacctaa tagtcctact 22440 gggcgctctt ttattgaaga tcttctcttt acaagtgtag aatctgttgg attaccaact 22500 gatgaagagt ataaaaagtg tacagcagga cctttaggtt ttgttaaaga ccttgtttgt 22560 gctagagagt ataatggttt gctcgttctg cctcctatta ttactgcgga aatgcaaacc 22620 atgtatacta gttctttagt agcctctatg gctttaggtg gcattactgc agctggtgct 22680 ataccttttg ctacacaact gcaggccaga attaaccatt tgggtattac taattctctt 22740 ttgttgaaaa accaagaaaa aattgctgct tcctttaata aggccatcgg tcatatgcag 22800 gaagggttta aaagtacttc tctagcatta caacagattc aagatgttgt taataaacag 22860 agttctattc ttacagagac tatgcaatca cttaataaaa attttggtgc tatttcctct 22920 gtaattcaag acatttacca gcaactagat gctattcagg cagatgctca ggttgatcgt 22980 cttattaacag gtagactctc ttcactatct gttttagctt ctgctaaaca ggcagagtat 23040 catagagtgt cacaacagcg tgagttggcc actcagaaaa ttaatgagtg tgttaagtct 23100 cagtctaata ggtattcatt ttgtggtaat ggtagacatg ttctaaccat accacagaat 23160 gcacccaatg gcatagtgtt tatacacttt acatacactc cagagagttt tgttaatgtt 23220 acggcaatag tagggttttg cgtaaaccca gctaatgcta gtcattatgc aatagtgcct 23280 gttaatggca ggggtgtttt tatagaagtt aatggtagtt actatatcac tgctcgtgat 23340 atgtatatgc caagagatat tactgcagga gacatagtca ctttgacttc ttgtcaagca 23400 aactatgtta atgtaaataa aaccgtcatt aacacttttg tggaagatga cgattttgat 23460 ttttatgatg aattgtcaaa atggtggaat gatactaagc atgagctacc agattttgat 23520 gaattcaatt ataccgttcc agttttaaat attagtaatg aaattgacag aattcaacag 23580 gttattcagg gattaaatga ttccctaata gaccttgaaa cactctcaat tctcaaaact 23640 tatattaaat ggccttggta tgtgtggctt gccattgcat tccttaccat tatttttt 23700 ctggtacttt gttggatatt tttcatgacc ggttgttgcg gttgttgttg tggatgcttt 23760 ggtatcatac cgttaatgag taagtgtggt aagaaatctt cttactacac gacttttgat 23820 aatgatgtgg taacttaaca atacagacct aaaaagtctg tttaatgatt aaaagtccca 23880 catcttttct aatattatta attcttcttt ggtgtaaact tgcattaagt tgttttaaag 23940 agtgtgttat aacactccag caactagtac aaattttact ccaaattatt aatagtaact 24000 tacaatctag acttctgctt tggcacagtc tagactaatg ttagattttg aagcaattat 24060 tgaaactggt cagcaaataa ctcaacaaat tagtttctat ttacagcata tttcaagggt 24120 gctaagtact gaattatttg acccctttga agtttgtgtt tacagaggag gtaattgttg 24180 ggagttagag tcagctgacg agttttcagg tgatgacgaa tatattgagt agatcgctag aggagacgg aagttttcta acagcggttt acgtgttttt aggtttt gcactttatc 24300. tactaggtag agcgcttcaa gcttttgtac aagcggctga cgcttgttgt cttttttggt attackgggt agtagttcct ggagccaagg gcacagcctt tgtttataat catacatatg gtaaaaaact taacaaaccg gagttagaaa cggttattgt taacgaattt ccaaaaaacg gttggaaata tggataatac catcaattgt actcttggta ctgaacaagc agttcagctt tttaaggat ataatctgtt tgtaactgca ttcctgttgt ttttaaccat actacttcag tatggatacg caactaggag caaggttatt tacatactga aaatgatagt gttatggtgc ttttggcccc ttaacattgc agtagtgta atctcatgta tatacccacc aaacacagga ggtcttgtcg cagcgataat tcttacagtg tttgcgtgtc tttcttttat aggttattgg atccagagta ttagactttt tagcggtgc aggtcatggt ggtcatttaa ccccgaatct aatgccgtag gttcaatact cctaactaat ggtcaacaat gtaattttgc tatagagagt gtgccgatgg tgctttctcc tattataag aatggtgctc tttattgcga gggtcagtgg 24960 cttgctaaat gtgaaccaga ccacttgcct agagatatat ttgtagcac accggataga 25020 cgtaatatct atcgtatggt gcaaaatat actggtgacc aaagcggaag taagaaaagg 25080 tttgccacat ttgtctatgc aaagcagtca gtagatactg gcgagctaga aagtgtgtca 25140 gcagtaggag gtagtcttta cacataaatg tgtgtgtgta gagagtattt aaaattattc 25200 tttgacagtg cctccgtttt aagagcgcgg aagagtatta tttttgagga tattatata 25260 aatcctcttt gttcatact ctccttcag gagttattat ttaaaaaca gttttccac 25320 tctttgtgc CAAAaacaat tgttgttaat ggtgtaacct ttcaggtaga caatggaaaa 25380 gtctactacg aaggagacc aattttcca aaaggttgtt gtagtttgtg gtccaattat 25440 aagaaagatt agaatatta aaccacctac aacacttatt tttacaatg gcgttttagg 25500 ttacaaacgc ttaacaata cggatgatga aatggctgac tagttttgga agagctttca 25560 tctcctgtta taaatcccta ttactaactc aattaagagt attagatagg ttaattttag 25620 atcacggacc caagcgcaca ttaacgtgtg ctaggcgagt gcttttagtt caattagatt 25680 tagtttatag gttggcttat acgcccaccc aatcgctggt atgaataata gtaaagataa 25740 tccttttcgc ggagcaatag caagaaaagc gcgaatttat ctgagaggag gattagattg 25800 tgtttacttt cttaacaaag caggacaagc agagccttgt cccgcgtgta cctccctagt 25860 attccaaggg aaaacttgtg aggaacacta ttataataac aatctttttgt catggcaagc 25920 ggtaaggcaa ctggaaagac agacgcccca gcgccagtca tcaaactagg aggaccaaag 25980 ccacctaaag ttggttcttc tggaaatgca tcatggtttc aaccgataaa ggccaagaag 26040 ctaaattcac ctgtgcctaa atttgacggt agtggtgttc ctgaaaatga aaatctcaag 26100 tcaagccagc aacatggata ctggagacgc caacacaggt ttaagcctgg caaaggtgga 26160 agaaaaccag tccctgatgc ttggtacttt tactacactg gaacaggacc ggccgccgac 26220 ctgaattggg gtgaaactca agatggtata gtgtgggttg ctgcaaaggg tgctgatact 26280 aaatctagat siaccagggg taaagggat cctgataagt ttgaccaata cccactacga 26340 ttctcagatg gaggaccgga tggtaatttc cgttgggact tcataccaat aaatcgtggt 26400 aggagtggga gatcacagc agcttcatca gcagcatcta gtagagcacc atctcgtgag 26460 gggtcacgtg gacgtagaag cggagttgaa gatgatctta tagctcgcgc agcaagatt 26520 attackaggacc agcaaagaa gggtgcgcgc attack ctaggctga tgaatggct 26580 catcgccgct attgcaagcg cactatccca cctggttata aggttgagca agtatttggt 26640 ccccgtacta aagtagga aggaatttt ggtgatgaca agatgaatga ggaggtgtt 26700 aaggatgggc gtgttacggc aatgctcaac ctagtcccta gcagtcatgc ttgtcttttt 26760 ggaagtaggg tgacgcccaa actgcagcca gatggtcttc acctgagatt tgaatttact 26820 actgtggtgt cacgtgatga tccgcagtttt gataattatg tgaaaatttg tgatcagtgt 26880 gtcgatggtg tagggacgcg tccaaggac gatgaatcga gaccaagctc acgcccaaat 26940 tcaagacctg caactagagg aaattctcca gcgccgagac aacagcgccc aaagaaggag 27000 aaaaagccca agaagcagga tgatgaagta gataaggcat tgacctcaga tgaggagagg 27060 aacaatgcac agctggaatt tgatgatgaa cccaaggtga ttaactgggg ggactctgca 27120 ctaggtgaaa atgaactttg attaacataa tggacttgct gcatttgctg tcacattttg 27180 ttaaatatta ttttgtgtt ttactatcaa ttattacagg tattgattgt gattatgttc 27240 aatacttaag cttcttctgg ttgctttttg cttgttgtat tgttgctgtg cttttatta 27300 ttgtgattct cattagtttg ctttatcgta gaaattcaat agtaagagtt aaggaagata 27360 ggcatgtagc ttagcaccta catgtctatc gccagggaaa tgtctaatct gtctacttag 27420 tagcctggaa acgaacggta gacccttaga ttttaattta gtttaatttt tagtttagtt 27480 taagttagtt tagagtaggt ataaagatgc cagtgccggg gccacgcgta gtacgaccga 27540 gggtacagca ctaggacgcc cactagggga agagctaaat tttagtttaa gttaagttta 27600 attggctaaa tatagttaaa atttataggc tagtatag ttagagcaaa aaaaaaaa 27659 <210> 2 <211> 1155 <212> PRT <213> Avian infectious bronchitis virus <400> 2 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> 3 <211> 409 <212> PRT <213> Avian infectious bronchitis virus <400> 3 Met Ala Ser Gly Lys Ala 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 Pro Ile Lys Ala Lys Lys Leu Asn Ser Pro Val 35 40 45 Pro Lys Phe Asp Gly Ser Gly Val Pro Glu Asn Glu Asn Leu Lys Ser 50 55 Ser Gln Gln His Gly Tyr Trp Arg Arg Gln His Arg Phe Lys Pro Gly 65 70 75 80 Lys Gly Gly Arg Lys Pro Val Pro Asp Ala Trp Tyr Phe Tyr Tyr Thr 85 90 95 Gly Thr Gly Pro Ala Ala Asp Leu Asn Trp Gly Glu Thr Gln Asp Gly 100 105 110 Ile Val Trp Val Ala 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 Glu Gly Ser Arg Gly Arg Arg Ser Gly Val 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 Ala 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 Glu 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 Val 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 Ser 325 330 335 Arg Pro Lys Ser Arg Pro Asn Ser Arg Pro Ala Thr Arg Gly Asn Ser 340 345 350 Pro Ala Pro Arg Gln Gln Arg Pro Lys Lys Glu Lys Lys Pro 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> 4 <211> 94 <212> PRT <213> Avian infectious bronchitis virus <400> 4 Met Thr Asn Ile Leu Ser Arg Ser Leu Glu Glu Asn Gly Ser Phe Leu 1 5 10 15 Thr Ala Val Tyr Val Phe Leu Gly Phe Leu Ala Leu Tyr Leu Leu Gly 20 25 30 Arg Ala Leu Gln Ala Phe Val Gln Ala Ala Asp Ala Cys Cys Leu Phe 35 40 45 Trp Tyr Thr Trp Val Val Val Pro Gly Ala Lys Gly Thr Ala Phe Val 50 55 60 Tyr Asn His Thr Tyr Gly Lys Lys Leu Asn Lys Pro Glu Leu Glu Thr 65 70 75 80 Val Ile Val Asn Glu Phe Pro Lys Asn Gly Trp Lys Tyr Gly 85 90 <210> 5 <211> 224 <212> PRT <213> Avian infectious bronchitis virus <400> 5 Met Asp Asn Thr Ile Asn Cys Thr Leu Gly Thr Glu Gln Ala Val Gln 1 5 10 15 Leu Phe Lys Glu Tyr Asn Leu Phe Val Thr Ala Phe Leu Leu Phe Leu 20 25 30 Thr Ile Leu Leu Gln Tyr Gly Tyr Ala Thr Arg Ser Lys Val 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 Ile 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 Ala Leu Tyr Cys Glu Gly Gln Trp Leu Ala Lys 145 150 155 160 Cys Glu Pro Asp His Leu Pro Arg 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 Ser 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 Ser Ala Val Gly Gly Ser Leu Tyr 210 215 220 <210> 6 <211> 1162 <212> PRT <213> Avian infectious bronchitis virus <400> 6 Met Leu Val Thr Pro Leu Leu Leu Val Thr Leu Leu Cys Ala Leu Cys 1 5 10 15 Ser Ala Ala Leu Tyr Asp Ser Ser Ser Tyr Val Tyr Tyr Tyr Gln Ser 20 25 30 Ala Phe Arg Pro Pro Asp Gly Trp His Leu His Gly Gly Ala Tyr Ala 35 40 45 Val Val Asn Ile Ser Ser Glu Ser Asn Asn Ala Gly Ser Ser Ser Gly 50 55 60 Cys Thr Val Gly Ile Ile His Gly Gly Arg Val Val Asn Ala Ser Ser 65 70 75 80 Ile Ala Met Thr Ala Pro Ser Ser Gly Met Ala Trp Ser Ser Ser Gln 85 90 95 Phe Cys Thr Ala Tyr Cys Asn Phe Ser Asp Thr Thr Val Phe Val Thr 100 105 110 His Cys Tyr Lys His Gly Gly Cys Pro Ile Thr Gly Met Leu Gln Gln 115 120 125 His Ser Ile Arg Val Ser Ala Met Lys Asn Gly Gln Leu Phe Tyr Asn 130 135 140 Leu Thr Val Ser Val Ala Lys Tyr Pro Thr Phe Lys Ser Phe Gln Cys 145 150 155 160 Val Asn Asn Leu Thr Ser Val Tyr Leu Asn Gly Asp Leu Val Tyr Thr 165 170 175 Ser Asn Glu Thr Thr Asp Val Thr Ser Ala Gly Val Tyr Phe Lys Ala 180 185 190 Gly Gly Pro Ile Thr Tyr Lys Val Met Arg Glu Val Arg Ala Leu Ala 195 200 205 Tyr Phe Val Asn Gly Thr Ala Gln Asp Val Ile Leu Cys Asp Gly Ser 210 215 220 Pro Arg Gly Leu Leu Ala Cys Gln Tyr Asn Thr Gly Asn Phe Ser Asp 225 230 235 240 Gly Phe Tyr Pro Phe Thr Asn Ser Ser Leu Val Lys Gln Lys Phe Ile 245 250 255 Val Tyr Arg Glu Asn Ser Val Asn Thr Thr Phe Thr Leu His Asn Phe 260 265 270 Thr Phe His Asn Glu Thr Gly Ala Asn Pro Asn Pro Ser Gly Val Gln 275 280 285 Asn Ile Gln Thr Tyr Gln Thr Gln Thr Ala Gln Ser Gly Tyr Tyr Asn 290 295 300 Phe Asn Phe Ser Phe Leu Ser Ser Phe Val Tyr Lys Glu Ser Asn Phe 305 310 315 320 Met Tyr Gly Ser Tyr His Pro Ser Cys Asn Phe Arg Leu Glu Thr Ile 325 330 335 Asn Asn Gly Leu Trp Phe Asn Ser Leu Ser Val Ser Ile Ala Tyr Gly 340 345 350 Pro Leu Gln Gly Gly Cys Lys Gln Ser Val Phe Ser Gly Arg Ala Thr 355 360 365 Cys Cys Tyr Ala Tyr Ser Tyr Gly Gly Pro Leu Leu Cys Lys Gly Val 370 375 380 Tyr Ser Gly Glu Leu Asp His Asn Phe Glu Cys Gly Leu Leu Val Tyr 385 390 395 400 Val Thr Lys Ser Gly Gly Ser Arg Ile Gln Thr Ala Thr Glu Pro Pro 405 410 415 Val Ile Thr Gln His Asn Tyr Asn Asn Ile Thr Leu Asn Thr Cys Val 420 425 430 Asp Tyr Asn Ile Tyr Gly Arg Thr Gly Gln Gly Phe Ile Thr Asn Val 435 440 445 Thr Asp Ser Ala Val Ser Tyr Asn Tyr Leu Ala Asp Ala Gly Leu Ala 450 455 460 Ile Leu Asp Thr Ser Gly Ser Ile Asp Ile Phe Val Val Gln Ser Glu 465 470 475 480 Tyr Gly Leu Asn Tyr Tyr Lys Val Asn Pro Cys Glu Asp Val Asn Gln 485 490 495 Gln Phe Val Val Ser Gly Gly Lys Leu Val Gly Ile Leu Thr Ser Arg 500 505 510 Asn Glu Thr Gly Ser Gln Leu Leu Glu Asn Gln Phe Tyr Ile Lys Ile 515 520 525 Thr Asn Gly Thr Arg Arg Phe Arg Arg Ser Ile Thr Glu Ser Val Glu 530 535 540 Asn Cys Pro Tyr Val Ser Tyr Gly Lys Phe Cys Ile Lys Pro Asp Gly 545 550 555 560 Ser Ile Ala Thr Ile Val Pro Lys Gln Leu Glu Gln Phe Val Ala Pro 565 570 575 Leu Leu Asn Val Thr Glu Asn Val Leu Ile Pro Asn Ser Phe Asn Leu 580 585 590 Thr Val Thr Asp Glu Tyr Ile Gln Thr Arg Met Asp Lys Val Gln Ile 595 600 605 Asn Cys Leu Gln Tyr Ile Cys Gly Asn Ser Leu Glu Cys Arg Asn Leu 610 615 620 Phe Gln Gln Tyr Gly Pro Val Cys Asp Asn Ile Leu Ser Val Val Asn 625 630 635 640 Ser Val Gly Gln Lys Glu Asp Met Glu Leu Leu Asn Phe Tyr Ser Ser 645 650 655 Thr Lys Pro Ala Gly Phe Asn Thr Pro Val Leu Ser Asn Val Ser Thr 660 665 670 Gly Glu Phe Asn Ile Thr Leu Phe Leu Thr Thr Pro Ser Ser Pro Arg 675 680 685 Arg Arg Ser Phe Ile Glu Asp Leu Leu Phe Thr Ser Val Glu Ser Val 690 695 700 Gly Leu Pro Thr Asp Asp Ala Tyr Lys Asn Cys Thr Ala Gly Pro Leu 705 710 715 720 Gly Phe Leu Lys Asp Leu Ala Cys Ala Arg Glu Tyr Asn Gly Leu Leu 725 730 735 Val Leu Pro Pro Ile Ile Thr Ala Glu Met Gln Thr Leu Tyr Thr Ser 740 745 750 Ser Leu Val Ala Ser Met Ala Phe Gly Gly Ile Thr Ala Ala Gly Ala 755 760 765 Ile Pro Phe Ala Thr Gln Leu Gln Ala Arg Ile Asn His Leu Gly Ile 770 775 780 Thr Gln Ser Leu Leu Leu Lys Asn Gln Glu Lys Ile Ala Ala Ser Phe 785 790 795 800 Asn Lys Ala Ile Gly His Met Gln Glu Gly Phe Arg Ser Thr Ser Leu 805 810 815 Ala Leu Gln Gln Ile Gln Asp Val Val Asn Lys Gln Ser Ala Ile Leu 820 825 830 Thr Glu Thr Met Ala Ser Leu Asn Lys Asn Phe Gly Ala Ile Ser Ser 835 840 845 Val Ile Gln Glu Ile Tyr Gln Gln Leu Asp Ala Ile Gln Ala Asn Ala 850 855 860 Gln Val Asp Arg Leu Ile Thr Gly Arg Leu Ser Ser Leu Ser Val Leu 865 870 875 880 Ala Ser Ala Lys Gln Ala Glu Tyr Ile Arg Val Ser Gln Gln Arg Glu 885 890 895 Leu Ala Thr Gln Lys Ile Asn Glu Cys Val Lys Ser Gln Ser Ile Arg 900 905 910 Tyr Ser Phe Cys Gly Asn Gly Arg His Val Leu Thr Ile Pro Gln Asn 915 920 925 Ala Pro Asn Gly Ile Val Phe Ile His Phe Ser Tyr Thr Pro Asp Ser 930 935 940 Phe Val Asn Val Thr Ala Ile Val Gly Phe Cys Val Lys Pro Ala Asn 945 950 955 960 Ala Ser Gln Tyr Ala Ile Val Pro Ala Asn Gly Arg Gly Ile Phe Ile 965 970 975 Gln Val Asn Gly Ser Tyr Tyr Ile Thr Ala Arg Asp Met Tyr Met Pro 980 985 990 Arg Ala Ile Thr Ala Gly Asp Ile Val Thr Leu Thr Ser Cys Gln Ala 995 1000 1005 Asn Tyr Val Ser Val Asn Lys Thr Val Ile Thr Thr Phe Val Asp 1010 1015 1020 Asn Asp Asp Phe Asp Phe Asn Asp Glu Leu Ser Lys Trp Trp Asn 1025 1030 1035 Asp Thr Lys His Glu Leu Pro Asp Phe Asp Lys Phe Asn Tyr Thr 1040 1045 1050 Val Pro Ile Leu Asp Ile Asp Ser Glu Ile Asp Arg Ile Gln Gly 1055 1060 1065 Val Ile Gln Gly Leu Asn Asp Ser Leu Ile Asp Leu Glu Lys Leu 1070 1075 1080 Ser Ile Leu Lys Thr Tyr Ile Lys Trp Pro Trp Tyr Val Trp Leu 1085 1090 1095 Ala Ile Ala Phe Ala Thr Ile Ile Phe Ile Leu Ile Leu Gly Trp 1100 1105 1110 Val Phe Phe Met Thr Gly Cys Cys Gly Cys Cys Cys Gly Cys Phe 1115 1120 1125 Gly Ile Met Pro Leu Met Ser Lys Cys Gly Lys Lys Ser Ser Tyr 1130 1135 1140 Tyr Thr Thr Phe Asp Asn Asp Val Val Thr Glu Gln Tyr Arg Pro 1145 1150 1155 Lys Lys Ser Val 1160 <210> 7 <211> 1075 <212> PRT <213> Avian infectious bronchitis virus <400> 7 Ala Leu Tyr Asp Ser Ser Ser Tyr Val Tyr Tyr Tyr Gln Ser Ala Phe 1 5 10 15 Arg Pro Pro Asp Gly Trp His Leu His Gly Gly Ala Tyr Ala Val Val 20 25 30 Asn Ile Ser Ser Glu Ser Asn Asn Ala Gly Ser Ser Ser Gly Cys Thr 35 40 45 Val Gly Ile Ile His Gly Gly Arg Val Val Asn Ala Ser Ser Ile Ala 50 55 60 Met Thr Ala Pro Ser Ser Gly Met Ala Trp Ser Ser Ser Gln Phe Cys 65 70 75 80 Thr Ala Tyr Cys Asn Phe Ser Asp Thr Thr Val Phe Val Thr His Cys 85 90 95 Tyr Lys His Gly Gly Cys Pro Ile Thr Gly Met Leu Gln Gln His Ser 100 105 110 Ile Arg Val Ser Ala Met Lys Asn Gly Gln Leu Phe Tyr Asn Leu Thr 115 120 125 Val Ser Val Ala Lys Tyr Pro Thr Phe Lys Ser Phe Gln Cys Val Asn 130 135 140 Asn Leu Thr Ser Val Tyr Leu Asn Gly Asp Leu Val Tyr Thr Ser Asn 145 150 155 160 Glu Thr Thr Asp Val Thr Ser Ala Gly Val Tyr Phe Lys Ala Gly Gly 165 170 175 Pro Ile Thr Tyr Lys Val Met Arg Glu Val Arg Ala Leu Ala Tyr Phe 180 185 190 Val Asn Gly Thr Ala Gln Asp Val Ile Leu Cys Asp Gly Ser Pro Arg 195 200 205 Gly Leu Leu Ala Cys Gln Tyr Asn Thr Gly Asn Phe Ser Asp Gly Phe 210 215 220 Tyr Pro Phe Thr Asn Ser Ser Leu Val Lys Gln Lys Phe Ile Val Tyr 225 230 235 240 Arg Glu Asn Ser Val Asn Thr Thr Phe Thr Leu His Asn Phe Thr Phe 245 250 255 His Asn Glu Thr Gly Ala Asn Pro Asn Pro Ser Gly Val Gln Asn Ile 260 265 270 Gln Thr Tyr Gln Thr Gln Thr Ala Gln Ser Gly Tyr Tyr Asn Phe Asn 275 280 285 Phe Ser Phe Leu Ser Ser Phe Val Tyr Lys Glu Ser Asn Phe Met Tyr 290 295 300 Gly Ser Tyr His Pro Ser Cys Asn Phe Arg Leu Glu Thr Ile Asn Asn 305 310 315 320 Gly Leu Trp Phe Asn Ser Leu Ser Val Ser Ile Ala Tyr Gly Pro Leu 325 330 335 Gln Gly Gly Cys Lys Gln Ser Val Phe Ser Gly Arg Ala Thr Cys Cys 340 345 350 Tyr Ala Tyr Ser Tyr Gly Gly Pro Leu Leu Cys Lys Gly Val Tyr Ser 355 360 365 Gly Glu Leu Asp His Asn Phe Glu Cys Gly Leu Leu Val Tyr Val Thr 370 375 380 Lys Ser Gly Gly Ser Arg Ile Gln Thr Ala Thr Glu Pro Pro Val Ile 385 390 395 400 Thr Gln His Asn Tyr Asn Asn Ile Thr Leu Asn Thr Cys Val Asp Tyr 405 410 415 Asn Ile Tyr Gly Arg Thr Gly Gln Gly Phe Ile Thr Asn Val Thr Asp 420 425 430 Ser Ala Val Ser Tyr Asn Tyr Leu Ala Asp Ala Gly Leu Ala Ile Leu 435 440 445 Asp Thr Ser Gly Ser Ile Asp Ile Phe Val Val Gln Ser Glu Tyr Gly 450 455 460 Leu Asn Tyr Tyr Lys Val Asn Pro Cys Glu Asp Val Asn Gln Gln Phe 465 470 475 480 Val Val Ser Gly Gly Lys Leu Val Gly Ile Leu Thr Ser Arg Asn Glu 485 490 495 Thr Gly Ser Gln Leu Leu Glu Asn Gln Phe Tyr Ile Lys Ile Thr Asn 500 505 510 Gly Thr Arg Arg Phe Arg Arg Ser Ile Thr Glu Ser Val Glu Asn Cys 515 520 525 Pro Tyr Val Ser Tyr Gly Lys Phe Cys Ile Lys Pro Asp Gly Ser Ile 530 535 540 Ala Thr Ile Val Pro Lys Gln Leu Glu Gln Phe Val Ala Pro Leu Leu 545 550 555 560 Asn Val Thr Glu Asn Val Leu Ile Pro Asn Ser Phe Asn Leu Thr Val 565 570 575 Thr Asp Glu Tyr Ile Gln Thr Arg Met Asp Lys Val Gln Ile Asn Cys 580 585 590 Leu Gln Tyr Ile Cys Gly Asn Ser Leu Glu Cys Arg Asn Leu Phe Gln 595 600 605 Gln Tyr Gly Pro Val Cys Asp Asn Ile Leu Ser Val Val Asn Ser Val 610 615 620 Gly Gln Lys Glu Asp Met Glu Leu Leu Asn Phe Tyr Ser Ser Thr Lys 625 630 635 640 Pro Ala Gly Phe Asn Thr Pro Val Leu Ser Asn Val Ser Thr Gly Glu 645 650 655 Phe Asn Ile Thr Leu Phe Leu Thr Thr Pro Ser Ser Pro Arg Arg Arg 660 665 670 Ser Phe Ile Glu Asp Leu Leu Phe Thr Ser Val Glu Ser Val Gly Leu 675 680 685 Pro Thr Asp Asp Ala Tyr Lys Asn Cys Thr Ala Gly Pro Leu Gly Phe 690 695 700 Leu Lys Asp Leu Ala Cys Ala Arg Glu Tyr Asn Gly Leu Leu Val Leu 705 710 715 720 Pro Pro Ile Ile Thr Ala Glu Met Gln Thr Leu Tyr Thr Ser Ser Leu 725 730 735 Val Ala Ser Met Ala Phe Gly Gly Ile Thr Ala Ala Gly Ala Ile Pro 740 745 750 Phe Ala Thr Gln Leu Gln Ala Arg Ile Asn His Leu Gly Ile Thr Gln 755 760 765 Ser Leu Leu Leu Lys Asn Gln Glu Lys Ile Ala Ala Ser Phe Asn Lys 770 775 780 Ala Ile Gly His Met Gln Glu Gly Phe Arg Ser Thr Ser Leu Ala Leu 785 790 795 800 Gln Gln Ile Gln Asp Val Val Asn Lys Gln Ser Ala Ile Leu Thr Glu 805 810 815 Thr Met Ala Ser Leu Asn Lys Asn Phe Gly Ala Ile Ser Ser Val Ile 820 825 830 Gln Glu Ile Tyr Gln Gln Leu Asp Ala Ile Gln Ala Asn Ala Gln Val 835 840 845 Asp Arg Leu Ile Thr Gly Arg Leu Ser Ser Leu Ser Val Leu Ala Ser 850 855 860 Ala Lys Gln Ala Glu Tyr Ile Arg Val Ser Gln Gln Arg Glu Leu Ala 865 870 875 880 Thr Gln Lys Ile Asn Glu Cys Val Lys Ser Gln Ser Ile Arg Tyr Ser 885 890 895 Phe Cys Gly Asn Gly Arg His Val Leu Thr Ile Pro Gln Asn Ala Pro 900 905 910 Asn Gly Ile Val Phe Ile His Phe Ser Tyr Thr Pro Asp Ser Phe Val 915 920 925 Asn Val Thr Ala Ile Val Gly Phe Cys Val Lys Pro Ala Asn Ala Ser 930 935 940 Gln Tyr Ala Ile Val Pro Ala Asn Gly Arg Gly Ile Phe Ile Gln Val 945 950 955 960 Asn Gly Ser Tyr Tyr Ile Thr Ala Arg Asp Met Tyr Met Pro Arg Ala 965 970 975 Ile Thr Ala Gly Asp Ile Val Thr Leu Thr Ser Cys Gln Ala Asn Tyr 980 985 990 Val Ser Val Asn Lys Thr Val Ile Thr Thr Phe Val Asp Asn Asp Asp 995 1000 1005 Phe Asp Phe Asn Asp Glu Leu Ser Lys Trp Trp Asn Asp Thr Lys 1010 1015 1020 His Glu Leu Pro Asp Phe Asp Lys Phe Asn Tyr Thr Val Pro Ile 1025 1030 1035 Leu Asp Ile Asp Ser Glu Ile Asp Arg Ile Gln Gly Val Ile Gln 1040 1045 1050 Gly Leu Asn Asp Ser Leu Ile Asp Leu Glu Lys Leu Ser Ile Leu 1055 1060 1065 Lys Thr Tyr Ile Lys Trp Pro 1070 1075 <210> 8 <211> 1156 <212> PRT <213> Avian infectious bronchitis virus <400> 8 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> 9 <211> 1078 <212> PRT <213> Avian infectious bronchitis virus <400> 9 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> 10 <211> 1166 <212> PRT <213> Avian infectious bronchitis virus <400> 10 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> 11 <211> 1079 <212> PRT <213> Avian infectious bronchitis virus <400> 11 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> 12 <211> 1159 <212> PRT <213> Avian infectious bronchitis virus <400> 12 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> 13 <211> 1081 <212> PRT <213> Avian infectious bronchitis virus <400> 13 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> 14 <211> 1156 <212> PRT <213> Avian infectious bronchitis virus <400> 14 Met Leu Val Lys Ser Leu Phe Ile Val Thr Leu Leu Phe Ala Leu Cys 1 5 10 15 Ser Ala Ala Leu Phe Asp Asn Asn Gln Ala Val Tyr Tyr Tyr Gln Ser 20 25 30 Ala Phe Arg Pro Ser Ser Gly Trp His Lys His Gly Gly Ala Tyr Ala 35 40 45 Val Ala Asn Val Ser Leu Glu Tyr Ala Asn Ala Gly Ser Ser Thr His 50 55 60 Cys Thr Ala Gly Ala Ile Tyr Trp Ser Lys Asn Phe Thr Ala Ser Ser 65 70 75 80 Val Ala Met Thr Ala Pro Gly Thr Gly Met Ser Trp Ser Thr Ala Gln 85 90 95 Phe Cys Thr Ala His Cys Asn Phe Ser Asp Phe Thr Val Phe Val Thr 100 105 110 His Cys Tyr Lys Ser Gly Asp Val Cys Pro Leu Thr Gly Leu Ile Pro 115 120 125 Ser Gly Tyr Ile Arg Ile Ser Ala Met Thr Lys Gly Thr Thr Ser Leu 130 135 140 Phe Tyr Asn Leu Thr Val Pro Val Thr Lys Tyr Pro Lys Phe Lys Ser 145 150 155 160 Leu Gln Cys Val Asp Asn Phe Thr Ser Val Tyr Leu Asn Gly Asp Leu 165 170 175 Val Phe Thr Ser Asn Glu Thr Lys Asp Val Ser Ala Ala Gly Val His 180 185 190 Phe Lys Ala Gly Gly Pro Ile Thr Tyr Lys Val Met Glu Lys Val Asp 195 200 205 Val Leu Ala Tyr Phe Val Asn Gly Thr Ala Gln Asp Val Ile Leu Cys 210 215 220 Asp Asn Ser Pro Arg Gly Leu Leu Ala Cys Gln Tyr Asn Thr Gly Asn 225 230 235 240 Phe Ser Asp Gly Phe Tyr Pro Phe Thr Asn Ile Ser Leu Val Lys Glu 245 250 255 Lys Phe Ile Val Tyr Arg Glu Thr Ser Val Asn Thr Thr Leu Val Leu 260 265 270 Thr Asn Phe Thr Phe Thr Asn Val Ser Asn Ala Leu Pro Asn Thr Gly 275 280 285 Gly Val Asn Thr Ile Asn Ile Tyr Gln Thr Gln Thr Ala Gln Ser Gly 290 295 300 Cys Tyr Asn Phe Asn Phe Ser Phe Leu Ser Ser Phe Val Tyr Lys Gln 305 310 315 320 Ser Asp Phe Met Tyr Gly Ser Tyr His Pro Lys Cys Asp Phe Arg Pro 325 330 335 Glu Thr Ile Asn Asn Gly Leu Trp Phe Asn Ser Leu Ser Val Ser Leu 340 345 350 Ala Tyr Gly Pro Leu Gln Gly Gly Cys Lys Gln Ser Val Phe Ser Asn 355 360 365 Arg Ala Thr Cys Cys Tyr Ala Tyr Ser Tyr Asn Gly Pro Arg Leu Cys 370 375 380 Lys Gly Val Tyr Ile Gly Glu Leu Gln Gln Tyr Phe Glu Cys Gly Leu 385 390 395 400 Leu Val Tyr Val Thr Lys Ser Asp Gly Ser Arg Ile Gln Thr Arg Asn 405 410 415 Glu Pro Leu Val Leu Thr His His Asn Tyr Asn Asn Ile Thr Leu Asp 420 425 430 Arg Cys Val Glu Tyr Asn Ile Tyr Gly Arg Ser Gly Gln Gly Phe Ile 435 440 445 Thr Asn Val Thr Ala Ala Ala Ala Asn Tyr Asn Tyr Leu Ala Asp Gly 450 455 460 Gly Leu Ala Ile Leu Asp Thr Ser Gly Ala Ile Asp Ile Phe Val Val 465 470 475 480 Gln Gly Glu Tyr Gly Pro Asn Tyr Tyr Lys Val Asn Pro Cys Glu Asp 485 490 495 Val Asn Gln Gln Phe Val Val Ser Gly Gly Gly Ile Val Gly Val Leu 500 505 510 Thr Ser His Asn Glu Thr Gly Ser Gln Gln Leu Glu Asn Leu Phe Tyr 515 520 525 Val Lys Leu Thr Asn Ser Thr Arg Arg Thr Arg Arg Ser Thr Ile Ala 530 535 540 Asn Val Thr Thr Cys Pro Tyr Val Ser Tyr Gly Arg Phe Cys Ile Lys 545 550 555 560 Pro Asp Gly Leu Val Ser Glu Ile Val Pro Gln Glu Leu Asp Tyr Phe 565 570 575 Val Ala Pro Leu Leu Asn Val Thr Glu His Val Leu Ile Pro Asn Ser 580 585 590 Phe Asn Leu Thr Val Thr Asp Glu Tyr Ile Gln Thr Arg Met Glu Lys 595 600 605 Val Gln Ile Asn Cys Leu Gln Tyr Val Cys Gly Asn Ser Ile Glu Cys 610 615 620 Arg Asn Leu Phe Gln Gln Tyr Gly Pro Val Cys Asp Asn Ile Leu Ser 625 630 635 640 Ile Val Asn Ser Val Gly Gln Arg Glu Asp Met Glu Ser Leu Thr Phe 645 650 655 Tyr Ser Ser Thr Lys Pro Lys Gly Tyr Asn Thr Pro Ile Phe Ser Asn 660 665 670 Ile Ser Thr Gly Asp Phe Asn Ile Ser Leu Met Leu Thr Pro Pro Ser 675 680 685 Ser Pro Ser Gly Arg Ser Phe Ile Glu Asp Leu Leu Phe Thr Ser Val 690 695 700 Glu Thr Val Gly Leu Pro Thr Asp Ala Glu Tyr Lys Lys Cys Thr Ala 705 710 715 720 Gly Pro Leu Gly Thr Leu Lys Asp Leu Ile Cys Ala Arg Glu Tyr Asn 725 730 735 Gly Leu Leu Val Leu Pro Pro Ile Ile Thr Ala Asp Met Gln Thr Met 740 745 750 Tyr Thr Ala Ser Leu Val Gly Ala Met Ala Phe Gly Gly Ile Thr Ser 755 760 765 Ala Ala Ala Ile Pro Phe Ala Thr Gln Ile Gln Ala Arg Ile Asn His 770 775 780 Leu Gly Ile Thr Gln Ser Leu Leu Met Lys Asn Gln Glu Lys Ile Ala 785 790 795 800 Ala Ser Phe Asn Lys Ala Ile Gly His Met Gln Glu Gly Phe Arg Ser 805 810 815 Thr Ser Leu Ala Leu Gln Gln Ile Gln Asp Val Val Asn Lys Gln Ser 820 825 830 Ala Ile Leu Thr Glu Thr Met Asn Ser Leu Asn Lys Asn Phe Gly Ala 835 840 845 Ile Thr Ser Val Ile Gln Asp Ile Tyr Ala Gln Leu Asp Ala Ile Gln 850 855 860 Ala Asp Ala 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> 15 <211> 1078 <212> PRT <213> Avian infectious bronchitis virus <400> 15 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> 16 <211> 1169 <212> PRT <213> infectious diseases <400> 16 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> 17 <211> 1082 <212> PRT <213> Infectious bronchitis virus of poultry <400> 17 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> 18 <211> 11860 <212> DNA <213> Artificial sequence <220> <223> Plasmid with IBV <400> 18 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180 acdatagcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 240 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 300 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 360 tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt ggagatcggt acttcgcgaa 420 tgcgtcgaga tgagctctaa tacgactcac tatagggact taagtgtgat ataaatatat 480 atcatacata ctagccttgt gctagatttc caacttaaca aaacggactt aaatacctac 540 agttggtccc tataggtgtt ccattgcagt gcactttagt gccctggatg gcacctggcc 600 acctgtcagg tttttgttat taaaataata ttgttgctgg tatcactgct tgttttgccg 660 tgtctcactt tatacatccg ttgcttgggc tacctagtat ccagcgtcct actggcgttg 720 tggtcggttc gagtgcgaag aacctctggt tcatctagcg gtacgcgggt gtgtggaagt 780 agcgcttcag acgtaccggt tctgttgcgt gaaatacggg gtcacctccc cccacatacc 840 tctaagggct tttgagccta gcgttgggct acgttctcgc acaaggtcgg ctaacggcg 900 tttgtagggg gtagtgccaa acaacccctg aggtgacagg ttctggtggt gtttcgaaaa 960 caacaatgtg tgtgccgcat aatatgcgag taatgcattt tggagcagga agtgacaaag 1020 gagtggcccc aggtagcgct gttcttaggc agtggcttcc cgaaggtaca ctccttgtcg 1080 ataatgatat tggatatt gtatctgatg cacatgtctc tgtgctttca gattgcaata 1140 aatgtaaaac agagcacaag tttgatcttg tgatatctga tatgtataca gataatgatt 1200 caaagagaaa gcatgaaggc gttgtagcca ataacggcaa tgatgacgtc ttcatatacc 1260 tttcaaactt tcttcgtaac aatttagctc tgggaggcag ttttgccgta aaagtaacag 1320 agacaagttg gcatgagagt ttatatgaca ttgcacagga ttgtgcatgg tggacaatgt 1380 tttgtacagc agtgaatgct tcttcgtcag aagcattctt gattggtgtt aattattgg 1440 gtgcaagtga aaaggttaga gttagtggta aaaccctgca cgcaaattat atattttgga 1500 ggaattgtaa ttatttacaa acttcagcct atagtatatt tgacgttgct aagtttgatt 1560 tgaaattaaa agcaacgcca gttgtgaatt tgaaaactga acaaaagaca gacttagtct 1620 ttaatttaat taagtgtggt aagttactgg taagagatgt tggacaaacc gcttttacta 1680 gtgactcttt ggtatgcact atgtagtgct ttgctctatg ataataatta tattggtgat 1740 tttagatgta tccagcttgt gaactcaaac ggtgctaatg ttagtgctcc aagcattagc 1800 actgagaccg ttgaagtttc acaaggcctg gggacatatt atgtgttaga tcgagtttat 1860 ttaaatgcca cattattgct tactggttac tacccggtcg atggttctaa gtttagaaac 1920 ctcgctctta cgggaactaa ctcagttagc ttgtcgtggt ttcaaccacc ctatttaagt 1980 cagtttaatg atggcatatt tgcgaaggtg cagaacctta agacaagtac gccatcaggt 2040 gcaactgcat attttcctac tatagttata ggtagtttgt ttggctatac ttcctatacc 2100 gttgtaatag agccatataa tggtgttata atggcctcag tgtgccagta taccatttgt 2160 cagttacctt acactgattg taagcctaac actaatggta ataagcttat agggttttgg 2220 cacacggatg taaaaccccc aatttgtgtg ttaaagcgaa atttcacgct taatgttaat 2280 gctgatgcat tttattttca tttttaccaa catggtggta ctttttatgc gtactatgcg 2340 gataaaccct ccgctactac gtttttgttt agtgtatata ttggcgatat tttaacacag 2400 tattatgtgt tacctttcat ctgcaaccca acagctggta gcacttttgc tccgcgctat 2460 tgggttacac ctttggttaa gcgccaatat ttgtttaatt tcaaccagaa gggtgtcatt 2520 actagtgctg ttgattgtgc tagtagttat accagtgaaa taaaatgtaa gacccagagc 2580 atgttaccta gcactggtgt ctatgagtta tccggttata cggtccaacc agttggagtt 2640 gtataccggc gtgttgctaa cctcccagct tgtaatatag aggagtggct tactgctagg 2700 tcagtcccct cccctctcaa ctgggagcgt aagacttttc agaattgtaa ttttaattta 2760 agcagcctgt tacgttatgt tcaggctgag agtttgtttt gtaataatat cgatgcttcc 2820 aaagtgtatg gcaggtgctt tggtagtatt tcagttgata agtttgctgt accccgaagt 2880 aggcaagttg atttacagct tggtaactct ggatttctgc agactgctaa ttataagatt 2940 gatacagctg ccacttcgtg tcagctgcat tacaccttgc ctaagaataa tgtcaccata 3000 aacaaccata acccctcgtc ttggaatagg aggtatggct ttaatgatgc tggcgtcttt 3060 ggcaaaaacc aacatgacgt tgttacgct cagcaatgtt ttactgtaag atctagttat 3120 tgcccgtgtg ctcaaccgga catagttagc ccttgcacta ctcagactaa gcctaagtct 3180 gctttgtta atgtgggtga cattgtgaa ggcttaggtg ttttagaaga taattgtggc 3240 aatgctgatc cacataaggg ttgtatctgt gccaacaatt catttattgg atggtcacat 3300 gatacctgcc ttgttaatga tcgctgccaa atttttgcta atatattgtt aaatggcatt 3360 aatagtggta ccacatgttc cacagatttg cagttgccta atactgaagt ggttactggc 3420 atttgtgtca aatatgacct ctacggtatt actggacaag gtgtttttaa agaggttaag 3480 gctgactatt ataatagctg gcaaaccctt ctgtatgatg ttaatggtaa tttgaatggt 3540 tttcgtgatc ttaccactaa caagacttat acgataagga gctgttatag tggccgtgtt 3600 tctgctgcat ttcataaaga tgcacccgaa ccggctctgc tctatcgtaa tataaattgt 3660 agctatgttt ttagcaataa tatttcccgt gaggagaacc cacttaatta ctttgatagt 3720 tatttgggt gtgttgttaa tgctgataac cgcacggatg aggcgcttcc taattgtgat 3780 ctccgtatgg gtgctggctt atgcgttgat tattcaaaat cacgcagggc tcaccgatca 3840 gttctactg gctatcggtt aactacattt gagccataca ctccgatgtt agttaatgat 3900 agtgtccaat ccgttgatgg attatatgag atgcaaatac caaccaattt tactattggg 3960 caccatgagg agttcattca aactagatct ccaaaggtga ctatagattg tgctgcattt 4020 gtctgtggtg ataacactgc atgcaggcag cagttggttg agtatggctc tttctgtgtt 4080 aatgttaatg ccattcttaa tgaggttaat aacctcttgg ataatatgca actacaagtt 4140 gctagtgcat taatgcaggg tgttactata agttcgagac tgccagacgg catctcaggc 4200 cctatagatg acattaattt tagtcctcta cttggatgca taggttcaac atgtgctgaa 4260 gacggcaatg gacctagtgc aatccgaggg cgttctgcta tagaggatttt gttatttgac 4320 aaggtcaaat tatctgatgt tggctttgtc gaggcttata ataattgcac cggtggtcaa 4380 gaagttcgtg acctcctttg tgtacaatct tttaatggca tcaaagtatt acctcctgtg 4440 ttgtcagaga gtcagatctc tgctacaca accggtgcta ctgcggcagc tatgttccca 4500 ccgtggtcag cagctgccgg tgtgccattt agtttaagtg ttcaatatag aattaatggt 4560 ttaggtgtca ctatgaatgt gcttagtgag aaccaaaaga tgattgctag tgcttttaac 4620 aatgcgctgg gtgctatcca ggatgggttt gatgcaacca attctgcttt aggtaagatc 4680 cagtccgttg ttaatgcaaa tgctgaagca ctcaataact tactaaatca actttctaac 4740 aggttggtg ctattagtgc ttctttacaa gaaattctaa ctcggcttga ggctgtagaa 4800 gcaaaagccc agatagatcg tcttattaat ggcaggttaa ctgcacttaa tgcgtatata 4860 tccaagcaac ttagtgatag tacgcttatt aaagttagtg ctgctcaggc catagaaaag 4920 gtcaatgagt gcgttaagag ccaaaccacg cgtattaatt tctgtggcaa tggtaatcat 4980 atattatctc ttgtccagaa tgcgccttat ggcttatatt ttatacactt cagctatgtg 5040 ccaatatcct ttacaaccgc aaatgtgagt cctggacttt gcatttctgg tgatagagga ttagcaccta aagctggata ttttgttcaa gatgatggag aatggaagtt cacaggcagt tcatattact accctgaacc catcagat aaaaacagtg tcatattact tagttgcgca gtaaactaca caaaggcacc tgaagttttc ttgaacactt caatacctaa tccacccgac tttaaggagg agttagataa atggtttaag aatcagacgt ctattgcgcc tgatttatct ctcgatttcg agaagttaaa tgttactttg ctggacctga cgtatgagat gaacaggatt caggatgcaa ttaagaagtt aaatgagagc tacatcaacc tcaaggaagt tggcacatat gaaatgtatg tgaatggcc ttggtatgtg tggcttgcca ttgcattcct taccattatt tttattctgg tactttgttg gatattttc atgaccggtt gttgcggttg ttgttgtgga 5580 tgctttggta tcataccgtt aatgagtaag tgtggtaaga aatcttctta ctacacgact tttgataatg atgtggtaac ttaacaatac agacctaaaa agtctgttta atgattaaaa gtcccacatc ttttctaata ttattaattc ttctttggtg taaacttgca ttagttgtt ttaaagagtg tgttataaca ctccagcaac tagtacaaat tttactccaa attattaata 5820 gtaacttaca atctagactt ctgctttggc acagtctaga ctaatgttag attttgaagc 5880 aattattgaa actggtcagc aaataactca acaaattagt ttctatttac agcatatttc 5940 aagggtgcta agtactgaat tatttgaccc ctttgaagtt tgtgtttaca gaggaggtaa 6000 ttgttgggag ttagagtcag ctgacgagtt ttcaggtgat gacgaatata ttgagtagat 6060 cgctcgagga gaacggaagt tttctaacag cggtttacgt gtttttagga tttttagcac 6120 tttatctact aggtagagcg cttcaagctt ttgtacaagc ggctgacgct tgttgtcttt 6180 tttggtatac atgggtagta gttcctggag ccaagggcac agcctttgtt tataatcata 6240 catatggtaa aaaacttaac aaaccggagt tagaaacggt tattgttaac gaatttccaa 6300 aaaacggttg gaaatatgga taataccatc aattgtactc ttggtactga acaagcagtt 6360 cagcttttta aggaatataa tctgtttgta actgcattcc tgttgttttt aaccatacta 6420 cttcagtatg gatacgcaac taggagcaag gttatttaca tactgaaaat gatagtgtta 6480 tggtgctttt ggccccttaa cattgcagta ggtgtaatct catgtatata cccaccaaac 6540 acaggaggtc ttgtcgcagc gataattctt acagtgtttg cgtgtctttc ttttataggt 6600 tattggatcc agagtattag actttttaag cggtgcaggt catggtggtc attaacccc 6660 gaatctaatg ccgtaggttc aatactccta actaatggtc aacaatgtaa ttttgctata 6720 gagagtgtgc cgatggtgct ttctcctatt ataaagaatg gtgctcttta ttgcgagggt 6780 cagtggcttg ctaaatgtga accagaccac ttgcctagag atatatttgt atgcacaccg 6840 gatagacgta atatctatcg tatggtgcaa aatatactg gtgaccaaag cggaagtaag 6900 aaaaggtttg ccacatttgt ctatgcaaag cagtcagtag atactggcga gctagaaagt 6960 gtgtcagcag taggaggtag tctttacaca taaatgtgtg tgtgtagaga gtatttaaaa 7020 ttattctttg acagtgcctc cgttttaaga gcgcggaaga gtattatttt tgaggatatt 7080 aatataaatc ctctttgttt catactctcc tttcaggagt tattatttaa aaaacagttt 7140 ttccactctt ttgtgccaaa aacaattgtt gttaatggtg taacctttca ggtagacaat 7200 ggaaaagtct actacgaagg aagaccaatt ttccaaaaag gttgttgtag tttgtggtcc 7260 aattataaga aagattagaa taattaaacc acctacaaca cttattttta caaatggcgt 7320 tttaggttac aaacgcttaa caaatacgga tgatgaaatg gctgactagt tttggaagag 7380 ctttcatctc ctgttataaa tccctattac taactcaatt aagagtatta gataggttaa 7440 ttttagatca cggacccaag cgcacatta cgtgtgctag gcgagtgctt ttagttcaat 7500 tagatttagt ttataggttg gcttatacgc ccacccaatc gctggtatga ataataagtaa 7560 agataatcct tttcgcggag caatagcaag aaaagcgcga atttatctga gaggaggatt 7620 agattgtgtt tactttctta acaaagcagg acaagcagag ccttgtcccg cgtgtacctc 7680 cctagtattc caaggggaaaa cttgtgagga acactattat aataacaatc ttttgtcatg 7740 gcaagcggta aggcaactgg aaagacagac gccccagcgc cagtcatcaa actaggagga 7800 ccaaagccac ctaaagttgg ttcttctgga aatgcatcat ggtttcaacc gataaaggcc 7860 aagaagctaa attcacctgt gcctaaattt gacggtagtg gtgttcctga aaatgaaaat 7920 ctcaagtcaa gccagcaaca tggatactgg agacgccaac acaggtttaa gcctggcaaa 7980 ggtggaagaa aaccagtccc tgatgcttgg tacttttact acactggaac aggaccggcc 8040 gccgacctga attggggtga aactcaagat ggtatagtgt gggttgctgc aaagggtgct 8100 gatactaaat ctagatcaaa ccagggtaca agggatcctg ataagtttga ccaataccca 8160 ctacgattct cagatggagg accggatggt aatttccgtt gggacttcat accaataaat 8220 cgtggtagga gtgggagatc aacagcagct tcatcagcag catctagtag agcaccatct 8280 cgtgaggggt cacgtggacg tagaagcgga gttgaagatg atcttatagc tcgcgcagca 8340 aagattatac aggaccagca aaagaagggt gcgcgcatta ccaaggctaa ggctgatgaa 8400 atggctcatc gccgctattg caagcgcact atcccacctg gttaataaggt tgagcaagta 8460 tttggtcccc gtactaaagg taaggaagga aattttggtg atgacaagat gaatgaggaa 8520 ggtgttaagg atgggcgtgt tacggcaatg ctcaacctag tccctagcag tcatgcttgt 8580 ctttttggaa gtagggtgac gcccaaactg cagccagatg gttttcacct gagatttgaa 8640 ttactactg tggtgtcacg tgatgatccg cagtttgata attatgtgaa atttgtgat 8700 cagtgtgtcg atggtgtagg gacgcgtcca aaggacgatg aatcgagacc aaagtcacgc 8760 ccaaattca gacctgcaac taggaggaat tctccagcgc cgagacaa gcgcccaag 8820 aaggaaaaaaaggcagaaa gcaggatgat gaagtagata agcattgac ctcagatgag 8880 gagaggaaca atgcacagct ggaatttgat gatgaaccca agttgattta ctggggggac 8940 tctgcactag gtgaaaatga acttgatta acataatgga cttgctgcat ttgctgtcac 9000 attttgttaa atattattt tgtgttttac tatcattat tacaggtatt gattgtgatt 9060 atgttcaata cttaagcttc ttctggttgc ttttgctg tgtattgtt gctgtgcttt 9120 ttattattgt gattctcatt agtttgcttt atcgtagaaa ttcaatagta aggttaagg 9180 aagataggca tgtagcttag cacctacatg tctatcgcca gggaaatgtc taatctgtct 9240 acttagtagc ctggaaacga acggtagacc cttagatttt aatttagtttt aatttttagt 9300 ttagtttaag ttagtttaga gtaggtataa agatgccagt gccggggcca cgcgtagtac 9360 gaccgagggt acagcactag gacgcccact aggggaagag ctaaatttta gtttaagtta 9420 agtttaattg gctaaatata gttaaaattt ataggctagt atagagttag agcaaaaaaa 9480 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 9540 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaagcggccg catcggatgc cgggaccgac 9600 gagtgcagag gcgtgcaagc gagcttggcg taatcatggt catagctgtt tcctgtgtga 9660 aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc 9720 tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc 9780 cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc 9840 ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt 9900 cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca 9960 ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa 10020 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat 10080 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc 10140 cctggaagct cctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc 10200 gccttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt 10260 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac 10320 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg 10380 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca 10440 gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc 10500 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa 10560 accaccgctg gtagcggtgg ttttttgtt tgcaagcagc agattacgcg cagaaaaaaa 10620 ggatctcaag aagatccttt gatctttct acggggtctg acgctcagtg gaacgaaaac 10680 tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta 10740 attaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt 10800 taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc 10920 agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc 11160 agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc 11280 atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc 11520 agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc 11580 gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca 11640 cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt 11700 tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt 11760 ccgcgcacat ttccccgaaa agtgccacct gacgtctaag aaaccattat tatcatgaca 11820 ttaacctata aaaataggcg tatcacgagg ccctttcgtc 11860 <210> 19 <211> 3489 <212> DNA <213> Avian infectious bronchitis virus <400> 19 atgttggtaa cacctctttt actagtgact cttttgtgtg cactatgtag tgctgctttg 60 tatgactcga gttcttacgt gtactactac caaagtgcct tcagaccacc tgatggttgg 120 catttacatg ggggtgcgta tgcggttgtt aatatttcta gtgaatctaa taatgcaggc 180 tcttcatctg ggtgtactgt tggtattatt catggtggtc gtgttgttaa tgcttcttct 240 atagctatga cggcaccgtc atcaggtatg gcttggtcta gcagtcagtt ttgtactgca 360. tactgtaact tttcagatac tacagtgttt gttacacatt gttataaaca tggtgggtgt cctataactg gcatgcttca acagcattct atacgtgttt ctgctatgaa aaatggccag cttttctata atttaacagt tagtgtagct aagtacccta cttttaaatc atttcagtgt gttaataatt taacatccgt ataatttaaat ggtgatcttg tttacacctc taatgagacc acagatgtta catctgcagg tgtttatttt aaagctggtg gacctataac ttataaagtt atgagagaag ttagagccct ggcttatttt gttaatggta ctgcacaaga tgttattttg tgtgatgggt cacctagagg cttgttagca tgccagtata atactggcaa tttttcagat ggcttttatc cttttactaa tagtagttta gttaagcaga agtttattgt ctatcgtgaa aatagtgtta atactacttt tacgttacac aatttcactt ttcataatga gactggcgcc aacccaaatc ctagtggtgt ccagaatt caaacttacc aaacacaaac agctcagagt ggtttata attttaatttt ttccttttctg agtagttttg ttttaagga gtctaatttt atgtatggat cttatcaccc aagttgtaat tttagactag aaactattaa taatggtttg 1020 tggtttaatt cactttcagt tagtattgct tacggtcctc ttcaaggtgg ttgcaagcaa 1080 tctgtcttta gtggtagagc aacctgttgt tatgcttact catatggagg tcctttgctg 1140 tgtaaaggtg tttattcagg tgagttagat cataattttg aatgtggact gttagtttat 1200 gttactaaga gcggtggctc tcgtatacaa acagccactg aaccgccagt tataactcaa 1260 cacaattata ataatattac tttaaatact tgtgttgatt ataatatata tggcagaact 1320 ggccagggtt ttattactaa tgtaaccgac tcagctgtta gttataatta tctagcagac 1380 gcaggtttgg ctattttaga tacatctggt tccatagaca tctttgtcgt acaaagtgaa 1440 tatggtctta attattataa ggttaaccct tgcgaagatg tcaaccagca gtttgtagtt 1500 tctggtggta aattagtagg tattcttact tcacgtaatg agactggttc ccagcttctt 1560 gagaatcagt tttacatcaa aatcactaat ggaacacgtc gttttagacg ttctattact 1620 gaaagtgttg aaaattgcc ttatgttagt tatggtaagt tttgtataaa acctgatggc 1680 agtattgcca caatagtacc aaaacagtta gaacagttg tggcacctttt acttaatgtt 1740 actgaaaatg tgctcatacc tacagtttt aatttaacg ttacagatga gtacataca 1800 actcggatgg aaggtcca aattaattgc ctgcagtata ttgtgca ttctctggag 1860 tgcagaaatt tgttcaca atatggtcct gtttgcgaca acatattgtc tgtagtaaat 1920 agtgttggtc aaaagaga tatggaactt tgaatttct attctctac taagccggct 1980 ggttttaata caccagttct tagtaatgtt agcactggtg agtttaatat tactcttttt 2040 ttaacaacgc ctagtagtcc taggaggcgt tctttattg aagaccttct atttacaagt 2100 gttgaatctg ttggattacc aacagatgac gcatacaaaa attgcactgc aggtccttta 2160 ggctttctga aagaccttgc atgtgctcgt gatataatg gtttgcttgt gttgcctcct 2220 attataacag cagaaatgca aactttgtat acagctctc tagtagctc tatggctttt 2280 ggtggtatta ctgcagctgg tgctatacct ttgccacac aactgcaggc tagattaat 2340 cacttgggta ttacccagtc acttctttg aagaatcaag aaaaaattgc tgcttccttt 2400 aataaggcca tcggtcatat gcaggaaggt tttagaagta catctttagc attacaacaa 2460 attcaagatg ttgttaataa gcagagtgct attcttactg agactatggc atcacttaat 2520 aaaaatttg gtgccatttc ttctgtgatt caagaaatct accagcaact tgacgccata 2580 caagcaaatg ctcaagtgga tcgtcttata actggtagat tgtcatcact ttctgtttta 2640 gcatctgcta agcaggcgga gtatattaga gtgtcacaac agcgtgagtt agctactcag 2700 aagattaatg agtgtgttaa gtcacagtcc attaggtact ccttttgtgg taatggacga 2760 catgttttaa ccataccgca aaatgcacct aatggtatag tgtttataca cttttcttac 2820 actccagata gttttgttaa tgttactgca atagtgggtt tttgtgtaaa gccagctaat 2880 gctagtcagt atgcaatagt acccgctaat ggtaggggta tttttataca agttaatggt 2940 agttactaca tcactgcacg agatatgtat atgccaagag ctattactgc aggagatata 3000 gttacgctta cttcttgtca agcaaattat gtaagtgtaa ataagaccgt cattactaca 3060 ttcgtagaca atgatgattt tgattttaat gacgaattgt caaaatggtg gaatgatact 3120 aagcatgagc taccagactt tgacaaattc aattacacag tacctatact tgacattgat 3180 agtgaaattg atcgtattca aggcgttata cagggtctta atgactctct aatagacctt 3240 gaaaaacttt caatactcaa aacttatatt aagtggcctt ggtatgtgtg gctagccata 3300 gcttttgcca ctattatctt catcttaata ttaggatggg ttttcttcat gactgggtgt 3360 tgtggttgtt gttgtggatg ctttggcatt atgcctctaa tgagtaagtg tggtaagaaa 3420 tcttcttatt acacgacttt tgataacgat gtggtaactg aacaatacag acctaaaaag 3480 tctgtttaa 3489 <210> 20 <211> 3495 <212> DNA <213> Avian infectious bronchitis virus <400> 20 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 gattttatgt atgggtcata ccacccacat tgtaagttta gaccagagaa tattaat ggcttatggt ttaattcatt atctgtgtca cttacttacg gacccattca aggtggttgt 1080 aagcaatctg tttttagtaa tagagcaact tgttgctatg cttattctta tcaagggcct buy agggtgttta buy ctaacgcaat actttgaatg tggacttcta gtttacgtaa ctaagagtga tggctctcgt atacaaacta gaagtgaacc actggtgtta actcaata attack attack cattacttta fathergtgtg ttgagtata father agggttggtc aaggttttat tactatgta actgaagcaa ctgctaatta tagttatcta gcagatggtg gtttagctat tttagatacc tcaggagcca tagcatatt tgttgttcaa ggtgcatatg gtcttaatta ttataaggtt aatccctgtg aagatgttaa ccaacagttt gtagtgtctg gtggcaactt agttggcatt cttacatctc father aggttctgaa tctattgaga accagtttta catcaaactc actaacgga cacgtcgctc tagacgttct gttactggga atgttacaaa ttgcccttat gttagttatg gcaagttttg tataaaacca 1680. gatggttctt tatctataat agtaccacaa gaattagaac agtttgtggc gcctttattc 1740 aatgttactg agcatgtgct catacctgat agttttaatt taactgtcac agatgagtac 1800 atacaaactc gtatggataa ggttcaaatt atttgccttc agtatgtttg tggtaattct 1860 attgaatgca gaaagttgtt tcagcagtat ggacctgttt gtgaataat attgtctgtt 1920 gtaaatggtg taggtcaaag agaggatatg gaacttttaa gtttctattc ttctactaaa 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 tcctttaata aggccatcgg tcatatgcag gaagggttta aaagtacttc tctagcatta 2460 caacagattc aagatgttgt taataaacag agttctattc ttacagagac tatgcaatca 2520 cttaataaaa attttggtgc tatttcctct gtaattcaag acatttacca gcaactagat 2580 gctattcagg cagatgctca ggttgatcgt ctttatacag 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 aacacttttg tggaagatga cgattttgat ttttatgatg 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> 21 <211> 11320 <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 attcgcatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 300 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 360 tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt ggagatcggt acttcgcgaa 420 tgcgtcgaga tgagctctaa tacgactcac tatagggact taagtgtgat ataaatatat 480 atcatacata ctagccttgt gctagatttc caacttaaca aaacggactt aaatacctac 540 agttggtccc tataggtgtt ccattgcagt gcactttagt gccctggatg gcacctggcc 600 acctgtcagg tttttgttat taaataata ttgttgctgg tatcactgct tgttttgccg 660 tgtctcactt tatacatccg ttgcttgggc tacctagtat ccagcgtcct actggcgttg 720 tggtcggttc gagtgcgaag aacctctggt tcatctagcg gtacgcgggt gtgtggaagt 780 agcgcttcag acgtaccggt tctgttgcgt gaaatacggg gtcacctccc cccacatacc 840 tctaagggct tttgagccta gcgttgggct acgttctcgc acaaggtcgg ctaacggcg 900 960. tttgtagggg gtagtgccaa acaacccctg aggtgacagg ttctggtggt gtttcgaaaa caacaatgtg tgtgccgcat aatatgcgag taatgcattt tggagcagga agtgacaaag gagtggcccc aggtagcgct gttcttaggc agtggcttcc cgaaggtaca ctccttgtcg 1080 father tgtagattat gtatctgatg cacatgtctc tgtgctttca gattgcaata aatgtaaaac agagcacaag tttgatcttg tgatatctga tatgtataca gataatgatt caaagagaaa gcatgaaggc gttgtagcca ataacggcaa tgatgacgtc ttcatatacc tttcaaactt tcttcgtac aatttagctc tgggaggcag ttttgccgta aaagtaacag agacaagttg gcatgagagt ttatatgaca ttgcacagga ttgtgcatgg tggacaatgt tttgtacagc agtgaatgct tcttcgtcag aagcattctt gattggtgtt aattattgg gtgcaagtga aaaggttaga gttagtggta aaaccctgca cgcaaattat atattttgga ggaattgtaa ttatttacaa acttcagcct atagtatatt tgacgttgct aagtttgatt tgaattaaa agcaacgcca gttgtgaatt tgaaaactga acaaaagaca gacttagtct ttaatttaat taagtgtggt aagttactgg taagagatgt tggacaaacc gcttttacta 1680 gtgactcttt ggtatgcact atgtagtgct ttgctctatg ataataatac ttacgtttac 1740 tactaccaaa gtgcctttag gcctggtcca ggttggcacc tatatggggg tgcttatgca 1800 gtagataggg ttttaatga aaccaacaat gcaggcagtg catctgattg cactgctggt 1860 actttttatg aaagccataa tatttctgct tcttctgtag ccatgacagt accacataat 1920 ggtatgtctt ggtcagcttc acaattttgt acagctcatt gtaacttctc agactttaca 1980 gtgttcgtta cgcattgttt taaaaatcaa ctcggtagtt gtcccttgac aggtatgatt 2040 cctcagaatc atattcgtat ttctgctatg agagatggag ttttgtttta taacttaaca 2100 gttagcgtat ctaaataccc tagatttaaa tcgcttcaat gtgttagcaa ttctacatct 2160 gtctatgtaa atggtgacct tgttttcact tctaatgaaa cttcttacgt tacgggtgca 2220 ggcgtttatt ttaaaagtgg tgggcctgta acttataaag tttgaaaga agttaaagcc 2280 ctagcctact ttattaatgg taccgcacaa gaggttattt tatgtgataa ctcacctaga 2340 ggtttgcttg catgtcagta taacactggt aatttttcag atggattcta cccttttact 2400 aatcattctt tagttaagga taggtttatt gtatatcgag aaagtagcac taacactact 2460 ttaaagttaa ctaatttcag ttttactaat gtaagtaatg cttctcctaa ttcaggtggc 2520 gttgatactt tccaattata tcaaacaagt actgctcagg atggttatta taattttaat 2580 ttatcatttc tgagtagttt tgtgtataaa ccatctgatt ttatgtatgg gtcataccac 2640 ccacattgta agtttagacc agagaatatt aataatggct tatggtttaa ttcattatct 2700 gtgtcactta cttacggacc cattcaaggt ggttgtaagc aatctgtttt tagtaataga 2760 gcaacttgtt gctatgctta ttcttatcaa gggcctagta gatgtaaggg tgtttataga 2820 ggggagctaa cgcaatactt tgaatgtgga cttctagttt acgtaactaa gagtgatggc 2880 tctcgtatac aaactagaag tgaaccactg gtgttaactc aatataatta taacaacatt 2940 actttaaata agtgtgttga gtataatata tatggtaggg ttggtcaagg ttttattact 3000 aatgtaactg aagcaactgc taattatagt tatctagcag atggtggttt agctatttta 3060 gatacctcag gagccataga catatttgtt gttcaaggtg catatggtct taattattat 3120 aaggttaatc cctgtgaaga tgttaaccaa cagtttgtag tgtctggtgg caacttagtt 3180 ggcattctta catctcataa tgaaacaggt tctgaatcta ttgagaacca gttttacatc 3240 aaactcacta acggaacacg tcgctctaga cgttctgtta ctgggaatgt tacaaattgc 3300 ccttatgtta gttatggcaa gttttgtata aaaccagatg gttctttatc tataatagta 3360 ccacaagaat tagaacagtt tgtggcgcct ttattcaatg ttactgagca tgtgctcata 3420 cctgatagtt ttaatttaac tgtcacagat gagtacatac aaactcgtat ggataaggtt 3480 caaattattt gccttcagta tgtttgtggt aattctattg aatgcagaaa gttgtttcag 3540 cagtatggac ctgtttgtga tatatattg tctgttgtaa atggtgtagg tcaaagagag 3600 gatatggaac ttttaagttt ctattcttct actaaaccta gtggttacaa tacaccaatt 3660 tttaataatg ttagcactgg tgactttaat atttcgctcc tactaacacc acctaatagt 3720 cctactgggc gctcttttat tgaagatctt cctttacaa gtgtagaatc tgttggatta 3780 ccaactgatg aagagtataa aaagtgtaca gcaggacctt taggttgtt taagacctt 3840 gtttgtgcta gagagtataa tggtttgctc gttctgccctc ctattattac tgcggaatg 3900 caaaccatgt atactagttc tttagtagcc tctatggctt taggtggcat tactgcagct 3960 ggtgctatac cttttgctac acactgcag gccagaatta accattggg tattactaat 4020 tctcttttgt tgaaaaacca agaaaaaatt gctgctctct ttaataaggc catcggtcat 4080 atgcaggaag gttttaaag tacttctcta gcattacaac agattcaaga tgttgttaat 4140 aaacagagtt ctattcttac agagacttg caatcactta ataaaaattt tggtgctatt 4200 tcctctgtaa ttcagacat ttaccagcaa ctagatgcta ttcaggcaga tgctcaggtt 4260 gatcgtctta ttacaggtag actctcttca ctatctgttt tagctctgc taaacaggca 4320 gagtatcata gagtgtcaca acagcgtgag ttggccactc agaaaattaa tgagtgtgtt 4380 aagtctcagt ctaataggta ttcattttgt ggtaatgta gatagttct aaccatacca 4440 cagaatgcac ccaatggcat agtgtttata cactttacat acacccaga gagttttgtt 4500 aatgttacgg caatagtagg gttttgcgta aacccagcta atgctagtca ttatgcaata 4560 gtgcctgtta atggcagggg tgtttttata gaagttaatg gtagttacta tatcactgct 4620 cgtgatatgt atatgccaag agatattact gcaggagaca tagtcacttt gacttcttgt 4680 caagcaaact atgttaatgt aaataaaacc gtcattaaca cttttgtgga agatgacgat 4740 tttgattttt atgatgaatt gtcaaaatgg tggaatgata ctaagcatga gctaccagat 4800 tttgatgaat tcaattatac cgttccagtt ttaaatatta gtaatgaaat tgacagaatt 4860 caacaggtta ttcagggatt aaatgattcc ctaatagacc ttgaaacact ctcaattctc 4920 aaaacttata ttaaatggcc ttggtatgtg tggcttgcca ttgcattcct taccattatt 4980 tttattctgg tactttgttg gatatttttc atgaccggtt gttgcggttg ttgttgtgga 5040 tgctttggta tcataccgtt aatgagtaag tgtggtaaga aatcttctta ctacacgact 5100 tttgataatg atgtggtaac ttaacaatac agacctaaaa agtctgttta atgattaaaa 5160 gtcccacatc ttttctaata ttattaattc ttctttggtg taaacttgca ttaagttgtt 5220 ttaaagagtg tgttataaca ctccagcaac tagtacaaat tttactccaa attattaata 5280 gtaacttaca atctagactt ctgctttggc acagtctaga ctaatgttag attttgaagc 5340 aattattgaa actggtcagc aaataactca acaaattagt ttctatttac agcatatttc 5400 aagggtgcta agtactgaat tatttgaccc ctttgaagtt tgtgtttaca gaggaggtaa 5460 ttgttgggag ttagagtcag ctgacgagtt ttcaggtgat gacgaatata ttgagtagat 5520 cgctcgagga gaacggaagt tttctaacag cggtttacgt gtttttagga tttttagcac 5580 tttatctact aggtagagcg cttcaagctt ttgtacaagc ggctgacgct tgttgtcttt 5640 tttggtatac atgggtagta gttcctggag ccaagggcac agcctttgtt tataatcata 5700 catatggtaa aaaacttaac aaaccggagt tagaaacggt tattgttaac gaatttccaa 5760 aaaacggttg gaaatatgga taataccatc aattgtactc ttggtactga acaagcagtt 5820 cagcttttta aggaatataa tctgtttgta actgcattcc tgttgttttt aaccatacta 5880 cttcagtatg gatacgcaac taggagcaag gttatttaca tactgaaaat gatagtgtta 5940 tggtgctttt ggccccttaa cattgcagta ggtgtaatct catgtatata cccaccaaac 6000 acaggaggtc ttgtcgcagc gataattctt acagtgtttg cgtgtctttc ttttataggt 6060 tattggatcc agagtattag actttttaag cggtgcaggt catggtggtc attaacccc 6120 gaatctaatg ccgtaggttc aatactccta actaatggtc aacaatgtaa ttttgctata 6180 gagagtgtgc cgatggtgct ttctcctatt ataaagaatg gtgctcttta ttgcgagggt 6240 cagtggcttg ctaaatgtga accagaccac ttgcctagag atatatttgt atgcacaccg 6300 gatagacgta atatctatcg tatggtgcaa aatatactg gtgaccaaag cggaagtaag 6360 aaaaggtttg ccacatttgt ctatgcaaag cagtcagtag atactggcga gctagaaagt 6420 gtgtcagcag taggaggtag tctttacaca taaatgtgtg tgtgtagaga gtatttaaaa 6480 ttattctttg acagtgcctc cgttttaaga gcgcggaaga gtattatttt tgaggatatt 6540 aatataaatc ctctttgttt catactctcc tttcaggagt tattatttaa aaaacagttt 6600 ttccactctt ttgtgccaaa aacaattgtt gttaatggtg taacctttca ggtagacaat 6660 ggaaaagtct actacgaagg aagaccaatt ttccaaaaag gttgttgtag tttgtggtcc 6720 aattataaga aagattagaa taattaaacc acctacaaca cttattttta caaatggcgt 6780 tttaggttac aaacgcttaa caaatacgga tgatgaaatg gctgactagt tttggaagag 6840 ctttcatctc ctgttataaa tccctattac taactcaatt aagagtatta gataggttaa 6900 ttttagatca cggacccaag cgcacatta cgtgtgctag gcgagtgctt ttagttcaat 6960 tagatttagt ttataggttg gcttatacgc ccacccaatc gctggtatga ataataagtaa 7020 agataatcct tttcgcggag caatagcaag aaaagcgcga atttatctga gaggaggatt 7080 agattgtgtt tactttctta acaaagcagg acaagcagag ccttgtcccg cgtgtacctc 7140 cctagtattc caaggggaaaa cttgtgagga acactattat aataacaatc ttttgtcatg 7200 gcaagcggta aggcaactgg aaagacagac gccccagcgc cagtcatcaa actaggagga 7260 ccaaagccac ctaaagttgg ttcttctgga aatgcatcat ggtttcaacc gataaaggcc 7320 aagaagctaa attcacctgt gcctaaattt gacggtagtg gtgttcctga aaatgaaaat 7380 ctcaagtcaa gccagcaaca tggatactgg agacgccaac acaggtttaa gcctggcaaa 7440 ggtggaagaa aaccagtccc tgatgcttgg tacttttact acactggaac aggaccggcc 7500 gccgacctga attggggtga aactcaagat ggtatagtgt gggttgctgc aaagggtgct 7560 gatactaaat ctagatcaaa ccagggtaca agggatcctg ataagtttga ccaataccca 7620 ctacgattct cagatggagg accggatggt aatttccgtt gggacttcat accaataaat 7680 cgtggtagga gtgggagatc aacagcagct tcatcagcag catctagtag agcaccatct 7740 cgtgaggggt cacgtggacg tagaagcgga gttgaagatg atcttatagc tcgcgcagca 7800 aagattatac aggaccagca aaagaagggt gcgcgcatta ccaaggctaa ggctgatgaa 7860 atggctcatc gccgctattg caagcgcact atcccacctg gttataaggt tgagcaagta 7920 tttggtcccc gtactaaagg taaggaagga aattttggtg atgacaagat gaatgaggaa 7980 ggtgttaagg atgggcgtgt tacggcaatg ctcaacctag tccctagcag tcatgcttgt 8040 ctttttggaa gtagggtgac gcccaaactg cagccagatg gttttcacct gagatttgaa 8100 ttactactg tggtgtcacg tgatgatccg cagtttgata attatgtgaa atttgtgat 8160 cagtgtgtcg atggtgtagg gacgcgtcca aaggacgatg aatcgagacc aaagtcacgc 8220 ccaaattcaa gacctgcaac taggaggaat tctccagcgc cgagacaa gcgcccaag 8280 aaggaaaaaaaggcagaaa gcaggatgat gaagtagata agcattgac ctcagatgag 8340 gagaggaaca atgcacagct ggaatttgat gatgaaccca agtgattta ctggggggac 8400 tctgcactag gtgaaaatga acttgatta acataatgga cttgctgcat tgctgtcac 8460 attttgttaa atattattt tgtgttttac tatcattat tacaggtatt gattgtgatt 8520 atgttcaata cttaagcttc ttctggttgc ttttgctg tgtattgtt gctgtgcttt 8580 ttattattgt gattctcatt agtttgcttt atcgtagaaa ttcatagta aggttaagg 8640 aagataggca tgtagcttag cacctacatg tctatcgcca gggaaatgtc taatctgtct 8700 acttagtagc ctggaaacga acggtagacc cttagatttt aatttagtttt aatttttagt 8760 ttagtttaag ttagtttaga gtaggtataa agatgccagt gccggggcca cgcgtagtac 8820 gaccgagggt acagcactag gacgcccact aggggaagag ctaaatttta gtttaagtta 8880 agtttaattg gctaaatata gttaaaattt ataggctagt atagagttag agcaaaaaaa 8940 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 9000 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaagcggccg catcggatgc cgggaccgac 9060 gagtgcagag gcgtgcaagc gagcttggcg taatcatggt catagctgtt tcctgtgtga 9120 aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc 9180 tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc 9240 cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc 9300 ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt 9360 cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca 9420 ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa 9480 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat 9540 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc 9600 cctggaagct cctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc 9660 gccttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt 9720 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac 9780 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg 9840 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca 9900 gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc 9960 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa 10020 accaccgctg gtagcggtgg ttttttgtt tgcaagcagc agattacgcg cagaaaaaaa 10080 ggatctcaag aagatccttt gatctttct acggggtctg acgctcagtg gaacgaaaac 10140 tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta 10200 attaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt 10260 taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc 10380 agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc 10620 agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc 10740 atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattgga aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc 11040 gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca 11100 cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt 11160 tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt 11220 ccgcgcacat ttccccgaaa agtgccacct gacgtctaag aaaccattat tatcatgaca 11280 ttaacctata aaaataggcg tatcacgagg ccctttcgtc 11320 <210> 22 <211> 11314 <212> DNA <213> Artificial sequence <220> <223> Plasmid with IBV <400> 22[[ID=2**]] 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 It should be noted that there seems to be a minor error in your provided text where the "2**" in the "[[ID=2**]] " should probably be a correct number or identifier. If this is a crucial part, please double-check and correct it for a more accurate translation.attcgcatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 300 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 360 tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt ggagatcggt acttcgcgaa 420 tgcgtcgaga tgagctctaa tacgactcac tatagggact taagtgtgat ataaatatat 480 atcatacata ctagccttgt gctagatttc caacttaaca aaacggactt aaatacctac 540 agttggtccc tataggtgtt ccattgcagt gcactttagt gccctggatg gcacctggcc 600 acctgtcagg tttttgttat taaataata ttgttgctgg tatcactgct tgttttgccg 660 tgtctcactt tatacatccg ttgcttgggc tacctagtat ccagcgtcct actggcgttg 720 tggtcggttc gagtgcgaag aacctctggt tcatctagcg gtacgcgggt gtgtggaagt 780 agcgcttcag acgtaccggt tctgttgcgt gaaatacggg gtcacctccc cccacatacc 840 tctaagggct tttgagccta gcgttgggct acgttctcgc acaaggtcgg ctaacggcg 900 tttgtagggg gtagtgccaa acaacccctg aggtgacagg ttctggtggt gtttcgaaaa 960 caacaatgtg tgtgccgcat aatatgcgag taatgcattt tggagcagga agtgacaaag gagtggcccc aggtagcgct gttcttaggc agtggcttcc cgaaggtaca ctccttgtcg 1080 father tgtagattat gtatctgatg cacatgtctc tgtgctttca gattgcaata aatgtaaaac agagcacaag tttgatcttg tgatatctga tatgtataca gataatgatt caaagagaaa gcatgaaggc gttgtagcca ataacggcaa tgatgacgtc ttcatatacc tttcaaactt tcttcgtac aatttagctc tgggaggcag ttttgccgta aaagtaacag agacaagttg gcatgagagt ttatatgaca ttgcacagga ttgtgcatgg tggacaatgt tttgtacagc agtgaatgct tcttcgtcag aagcattctt gattggtgtt aattattgg gtgcaagtga aaaggttaga gttagtggta aaaccctgca cgcaaattat atattttgga ggaattgtaa ttatttacaa acttcagcct atagtatatt tgacgttgct aagtttgatt tgaattaaa agcaacgcca gttgtgaatt tgaaaactga acaaaagaca gacttagtct ttaatttaat tagtgtggt aagttactgg tagtagtgt tggtaacacc tcttttacta gtgactcttt tgtgtgcact atgtagtgct gctttgtatg actcgagttc ttacgtgtac 1740 tactaccaaa gtgccttcag accacctgat ggttggcatt tacatggggg tgcgtatgcg 1800 gttgttaata tttctagtga atctaataat gcaggctctt catctgggtg tactgttggt 1860 attattcatg gtggtcgtgt tgttaatgct tcttctatag ctatgacggc accgtcatca 1920 ggtatggctt ggtctagcag tcagttttgt actgcatact gtaacttttc agatactaca 1980 gtgtttgtta cacattgtta taaacatggt gggtgtccta taactggcat gcttcaacag 2040 cattctatac gtgtttctgc tatgaaaaat ggccagcttt tctataattt aacagttagt 2100 gtagctaagt accctacttt taaatcattt cagtgtgtta ataatttaac atccgtatat 2160 ttaaatggtg atcttgttta cacctctaat gagaccacag atgttacatc tgcaggtgtt 2220 tattttaaag ctggtggacc tataacttat aaagttatga gagaagttag agccctggct 2280 tattttgtta atggtactgc acaagatgtt attttgtgtg atgggtcacc tagaggcttg 2340 ttagcatgcc agtataatac tggcaatttt tcagatggct tttatccttt tactaatagt 2400 agtttagtta agcagaagtt tattgtctat cgtgaaaata gtgttaatac tactttacg 2460 ttacacaatt tcactttca taatgact ggcgccaacc caatcctag tggtgtccag 2520 atattcaa cttaccaaac acaacagct cagagtggtt attataattt taatttttcc 2580 tttctgagta gttttgttta taggagtct aattttgt atggatctta tcaccagt 2640 tgtatttta gactagaac tattataat gttttgtggt ttattcact ttcagttagt 2700 attgcttacg gtccttca aggtggttgc aagcaatctg tcttagtgg tagagcaacc 2760 tgttgttatg cttactcata tggaggtcct tgctgtgta aaggtgttta ttcaggtg 2820 ttagatcata attttgaatg tggactgtta gtttagtta ctaagagcgg tggctctcgt 2880 atacaacag ccactgaacc gccagttata actcacaca attataata tattacttta 2940 atacttgtg ttgattataa tatatatggc agaactggcc agggttttat tactaatgta 3000 accgactcag ctgttagtta taattatcta gcagacgcag gttttggctat tttagataca 3060 tctggttcca tagacatctt tgtcgtacaa agtgaatatg gtctttatta ttataaggtt 3120 aacccttgcg aagatgtcaa ccagcagtttt gtagtttctg gtggtaaatt agtaggtatt 3180 cttacttcac gtaatgagac tggttcccag cttcttgaga atcagtttta catcaaatc 3240 actaatggaa cacgtcgttt tagacgttct attactgaaa gtgttgaaa ttgcccttat 3300 gttagttatg gtaagttttg tataaaacct gatggcagta ttgccacaat agtaccaaaa 3360 cagttagaac agttgtggc acctttactt aatgttactg aaatgtgct catacctaac 3420 agttttaatt taactgttac agatgagtac atacaactc ggatggataa ggtccaaatt 3480 aattgcctgc agtatattg tggcaatttct ctggagtgca gaaatttgtt tcacaat 3540 ggtcctgttt gcgacacacat attgtctgta gtaaatagtg ttggtcaaaa agagatatg 3600 gaacttttga atttctattc ttctaag ccggctggtt ttatacacc agttcttagt 3660 aatgttagca ctggtgagtt taatattact cttttta caacgcctag tagtcctaga 3720 aggcgttctt ttattgaga ccttctttt acagtgttg aatctgttgg attaccaaca 3780 gatgacgcat aaaaattg cactgcaggt cctttaggct tctgaaaga ccttgcatgt 3840 gctcgtgaat ataatggttt gcttgtgttg cctcctatta taacagcaga aatgcaaact 3900 ttgtatacaa gctctctagt agcttctatg gcttttggtg gtattactgc agctggtgct 3960 ataccttttg ccacacaact gcaggctaga attaatcact tgggtattac ccagtcactt 4020 cttttgaaga atcaagaaaa aattgctgct tcctttaata aggccatcgg tcatatgcag 4080 gaaggtttta gaagtacatc tttagcatta caacaaattc aagatgttgt taataagcag 4140 agtgctattc ttactgagac tatggcatca cttaataaaa attttggtgc catttcttct 4200 gtgattcaag aaatctacca gcaacttgac gccatacaag caaatgctca agtggatcgt 4260 cttataactg gtagattgtc atcactttct gttttagcat ctgctaagca ggcggagtat 4320 attagagtgt cacaacagcg tgagttagct actcagaaga ttaatgagtg tgttaagtca 4380 cagtccatta ggtactcctt ttgtggtaat ggacgacatg ttttaaccat accgcaaaat 4440 gcacctaatg gtatagtgtt tatacacttt tcttacactc cagatagttt tgttaatgtt 4500 actgcaatag tgggtttttg tgtaaagcca gctaatgcta gtcagtatgc aatagtaccc 4560 gctaatgta ggggtatttt tatacaagtt ...

Claims

1. An infectious bronchitis virus 4 / 91 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: SEQ ID NO: 6, 7, 8 and 9.

2. The infectious bronchitis virus 4 / 91 of claim 1, wherein the infectious bronchitis virus 4 / 91 is attenuated or recombinant.

3. An immunogenic composition comprising the infectious bronchitis virus 4 / 91 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 4 / 91 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 4 / 91 according to any one of claims 1 to 2 in the preparation of an immunogenic composition for treating or preventing infection caused by infectious bronchitis virus in poultry in need thereof.

7. Use of the infectious bronchitis virus 4 / 91 according to any one of claims 1 to 2 in 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 species.

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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