Attenuated infectious bronchitis viruses and vaccines comprising same

Abstract

The invention relates to the field of animal health. The present invention provides an attenuated infectious bronchitis virus (IBV). The attenuated infectious bronchitis virus can protect chickens from IBV infection.

Description

Cross-reference to related applications

[0001] This application claims priority to PCT / CN2023 / 117927, filed on September 11, 2023, entitled “Attenuated Infectious Bronchitis Virus and Vaccine Containing the Same”, the entire contents of which are incorporated herein by reference. Technical Field

[0002] This invention relates to the field of animal health. In particular, it relates to attenuated infectious bronchitis virus and attenuated live vaccines containing the same. The invention also relates to methods for preparing attenuated infectious bronchitis virus and methods for immunizing poultry by administering the vaccine. Background Technology

[0003] Infectious bronchitis (IB) is an acute and highly contagious infection caused by infectious bronchitis virus (IBV), and is one of the most serious viral infections in poultry. Multiple serotypes of IBV exist. Since the mid-1990s, new IBV serotypes have emerged in different regions. Different serotypes of IBV provide only partial cross-immunity, or even no cross-immunity at all. Since 1996, IB caused by serotype QX of IBV (IB-QX) has become an endemic disease in China. A vaccine against IB-QX needs to be developed. Summary of the Invention

[0004] In one aspect, the present invention provides an attenuated IBV, wherein the attenuated IBV comprises an S1 gene comprising a nucleotide sequence or a nucleotide sequence encoding an amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO:4; or the attenuated IBV comprises an S1 gene comprising a nucleotide sequence or a nucleotide sequence encoding an amino acid sequence selected from the following: SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6.

[0005] In one aspect, the present invention provides an attenuated IBV, wherein the attenuated IBV can be obtained by passage of the IB-QX K19 P7 strain to at least the 160th generation; preferably the 160th to the 200th generation; more preferably the 160th to the 180th generation; more preferably the 162nd to the 179th generation; more preferably the 162nd, 163rd, 164th, 165th, 166th, 167th, 168th, 169th, 170th, 171st, 172nd, 173rd, 174th, 175th, 176th, 177th, 178th or 179th generation; more preferably the 162nd, 171st or 179th generation, and wherein the IB-QX K19 P7 strain is deposited under accession number CCTCC NO: V202364.

[0006] In one aspect, the present invention provides the IB-QX K19-A P162 strain and its progeny, wherein the progeny of the IB-QX K19-A P162 strain is any attenuated progeny IBV having all the identifying characteristics of the deposited IB-QX K19-A P162 strain, and wherein the IB-QX K19-A P162 strain is deposited under accession number CCTCC NO: V202366.

[0007] In one aspect, the present invention provides the IB-QX K19-A P171 strain and its progeny, wherein the progeny of the IB-QX K19-A P171 strain is any attenuated progeny IBV having all the identifying characteristics of the deposited IB-QX K19-A P171 strain, and wherein the IB-QX K19-A P171 strain is deposited under accession number CCTCC NO: V202367.

[0008] In one aspect, the present invention provides the IB-QX K19-A P179 strain and its progeny, wherein the progeny of the IB-QX K19-A P179 strain is any attenuated progeny IBV having all the identifying characteristics of the deposited IB-QX K19-A P179 strain, and wherein the IB-QX K19-A P179 strain is deposited under accession number CCTCC NO: V202368.

[0009] In one aspect, the present invention provides an attenuated infectious bronchitis virus (IBV) selected from: the IB-QX K19-A P162 strain deposited under accession number CCTCC NO: V202366, any of the progeny of the IB-QX K19-A P162 strain comprising up to 5, 4, 3, 2, or 1 generations, the IB-QX K19-A P171 strain deposited under accession number CCTCC NO: V202367, any of the progeny of the IB-QX K19-A P171 strain comprising up to 5, 4, 3, 2, or 1 generations, the IB-QX K19-A P179 strain deposited under accession number CCTCC NO: V202368, and the IB-QX K19-A P179 strain comprising IB-QX K19-A P171. Any of the progeny of the IB-QX K19-A P179 strain at most 5, 4, 3, 2 or 1 generations.

[0010] In one aspect, the present invention provides a method for preparing attenuated IBV, comprising the following steps: passage an IBV strain in chicken embryos or chicken embryo-derived cells for up to 10 generations; more preferably, passage the IBV strain in chicken embryos or chicken embryo-derived cells for 5, 4, 3, 2 or 1 generations, wherein the IBV strain is selected from the IB-QX K19-AP162 strain deposited under accession number CCTCC NO: V202366, the IB-QX K19-A P171 strain deposited under accession number CCTCC NO: V202367, and the IB-QX K19-A P179 strain deposited under accession number CCTCC NO: V202368.

[0011] In one aspect, the present invention provides an immunogenic composition or live attenuated vaccine against IBV infection, comprising a therapeutically or preventively effective amount of the attenuated IBV of the present invention, or a therapeutically or preventively effective amount of attenuated IBV prepared by the method of the present invention.

[0012] In one aspect, the present invention provides a method for immunizing poultry, or a method for preventing or avoiding clinical signs caused by IBV in poultry, or a method for reducing ciliary arrest in poultry, wherein the method comprises administering to a subject a therapeutically or preventively effective amount of the immunogenic composition or attenuated live vaccine of the present invention, or a therapeutically or preventively effective amount of the immunogenic composition or attenuated live vaccine prepared by the method of the present invention.

[0013] In one aspect, the present invention provides the use of the attenuated IBV of the present invention in the preparation of immunogenic compositions or attenuated live vaccines for immunizing poultry; for preventing or mitigating clinical signs caused by IBV in poultry; or for alleviating ciliary stagnation in poultry.

[0014] The attenuated IBV strains of this invention have all been proven safe for chickens and exhibit good genetic stability, without virulence reversion after several passages. Furthermore, the attenuated IBV strains of this invention demonstrate excellent protective efficacy against IBV infection when administered via ocular or nasal drops or spray, thereby enabling convenient and large-scale vaccination in poultry farms. Additionally, the attenuated IBV strains of this invention exhibit excellent immunogenicity over a relatively wide passage range, ensuring that more passages can be used for the industrial production of IBV vaccines. Attached Figure Description

[0015] Figure 1 Sequence comparison results of the S1 gene between the IB-QX K19-A P162 strain and the IB-QX K19-A P171 strain.

[0016] Figure 2 Sequence comparison results of the S1 gene between the IB-QX K19-A P162 strain and the IB-QX K19-A P179 strain.

[0017] Figure 3 Sequence comparison results of the S1 gene between the IB-QX K19-A P171 strain and the IB-QX K19-A P179 strain.

[0018] Figure 4 Sequence comparison results of the S1 protein between the IB-QX K19-A P162 strain and the IB-QX K19-A P171 strain.

[0019] Figure 5 Sequence comparison results of the S1 protein between the IB-QX K19-A P162 strain and the IB-QX K19-A P179 strain.

[0020] Figure 6 Sequence comparison results of the S1 protein between the IB-QX K19-A P171 strain and the IB-QX K19-A P179 strain. Detailed Implementation

[0021] The invention discloses attenuated IBV, methods for preparing attenuated IBV, immunogenic compositions or attenuated live vaccines against IBV, methods for preparing immunogenic compositions or attenuated live vaccines against IBV, and methods for treating or preventing clinical signs caused by IBV in poultry.

[0022] Before describing various aspects of the invention, it must be noted that, as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural references unless the context clearly specifies otherwise. Thus, for example, a reference to “virus” refers to one or more viruses and their equivalents known to those skilled in the art, and so on.

[0023] The term “and / or” is intended to cover any combination of items connected by the term, equivalent to listing all combinations individually. For example, “A, B and / or C” covers “A”, “B”, “C”, “A and B”, “A and C”, “B and C”, and “A and B and C”.

[0024] The terms "include" and "including" have the same meaning as the terms "comprise" and "comprising" because these latter terms are "open-ended" transitional terms that do not limit the claims to the elements following those transitional terms. The term "composes of," while covered by the term "comprising," should be interpreted as a "closed-ended" transitional term that limits the claims to the elements following that transitional term.

[0025] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. While preferred methods, apparatuses, and materials are described herein, any methods and materials similar or equivalent to those described herein may be used in the practice or testing of this invention. All publications mentioned herein are incorporated by reference for the purpose of describing and disclosing viral strains, cell lines, vectors, and methods as reported in the publications that can be used in conjunction with this invention. Nothing herein should be construed as an admission that this invention is not entitled to any prior invention prior to such disclosure.

[0026] Attenuated infectious bronchitis virus (attenuated IBV) This invention provides an attenuated infectious bronchitis virus (IBV).

[0027] The term "infectious bronchitis virus (IBV)" refers to avian bronchitis virus, a protozoan belonging to the family Coronaviridae, order Nidovirales. IBV is an enveloped virus with a 27.6 kb positive-sense single-stranded RNA genome. The viral genome encodes structural proteins, including the spike protein (S), envelope protein (E), membrane protein (M), and nucleocapsid protein (N). Proteins S, E, and M form the viral envelope, while protein N, along with the viral RNA, forms the ribonucleoprotein core. Protein S is a dimeric or trimeric transmembrane protein that is proteasically cleaved into two subunits, S1 and S2. It is widely known and accepted in the art that the S1 protein is responsible for cell attachment and is the most important antigenic determinant of IBV. The S1 gene can also be used to identify IBV strains and their serotypes. As used herein, the term "S1 gene" refers to the polynucleotide encoding the S1 subunit of the IBV protein S.

[0028] Infectious bronchitis virus (IBV) primarily infects the upper respiratory tract epithelium of chickens and other poultry species, causing respiratory diseases. As used herein, unless otherwise stated, IBV is described as a highly virulent strain. The terms "infectious bronchitis virus," "IBV," and "IBV strain" are interchangeable in this invention.

[0029] IBV strains can be classified into different serotypes through serological studies and / or genetic and phylogenetic analysis of their S1 gene, with serotype QX being one of them. Therefore, the term "IB-QX" refers to IBV with serotype QX. Determining the serotype of an IBV strain is known in the art. For example, the serotype of the IBV strain of the present invention can be determined by comparing its S1 gene with the disclosed S1 gene of its corresponding serotype, which has already been determined.

[0030] In this invention, the K19 strain was isolated from the kidney tissue of chickens suspected of having IBV infection and then identified as a highly virulent IB-QX strain. As used herein, the term "IB-QX K19 strain" refers to this highly virulent IB-QX strain. The term "IB-QX K19 P7 strain" refers to another highly virulent strain obtained by passage of the IB-QX K19 strain to its 7th generation. The IB-QX K19 P7 strain is deposited under accession number CCTCC NO: V202364. The term "IB-QX K19 P8 strain" refers to another highly virulent strain obtained by passage of the IB-QX K19 strain to its 8th generation. The IB-QX K19 P8 strain is deposited under accession number CCTCC NO: V202365.

[0031] The term "attenuated IBV" refers to an IBV with reduced virulence compared to a virulent IBV strain. In this invention, an attenuated IBV is an IBV whose virulence has been reduced such that it does not cause clinical signs of IBV infection, but can induce an immune response in poultry. The term "reduced" means a reduction in virulence of at least 50%, preferably 60%, more preferably 70%, even more preferably 80%, even more preferably 90%, and even more preferably 95%, and most preferably 100%, compared to a virulent IBV strain.

[0032] Attenuation of virulent IBV strains can be achieved by passaged a virulent IBV strain at least a sufficient number of times, resulting in an IBV strain characterized by reduced virulence. In this invention, the IB-QX K19 strain is isolated and then passaged in chicken embryos to obtain several attenuated IBV strains with different passages. Methods for generating attenuated IBV strains by passage are known in the art. An exemplary method for passaged virulent IBV strains involves using chicken embryos as an environment conducive to viral replication. Chicken embryos are inoculated with a certain amount of virulent IBV strain via the allantoic cavity. The chicken embryos are incubated under suitable conditions, and then allantoic fluid is harvested from the embryos. At this point, the IBV strain has been passaged once (generation 1). The allantoic fluid harvested from the first passage is then inoculated into new chicken embryos at an appropriate dilution, which are incubated again, and then allantoic fluid is harvested from the second chicken embryo. At this point, the IBV strain has been passaged twice (generation 2). The virulence level of the IBV strain can be tested after each passage. This process is continued until an attenuated IBV strain is obtained. Example 1 illustrates an exemplary method for attenuating a highly virulent IBV strain through continuous passage.

[0033] As used herein, unless otherwise stated, the IB-QX K19 strain is used as the starting point for specifying the passage of the IB-QX strain. For example, the term "IB-QX K19-A P162 strain" refers to the 162nd generation of the IB-QX K19 strain. Thus, for example, the expression "passing the IB-QX K19 P7 strain to the 162nd generation" as used herein means that the passage begins with the IB-QX K19 P7 strain and continues until the 162nd generation of the IB-QX K19 strain. Therefore, unless otherwise stated, all passage numbers described herein are determined based on the IB-QX K19 strain. The IB-QX K19-A P162 strain is an attenuated IBV strain, and therefore the name "IB-QX K19-A P162 strain" includes the symbol "-A". The IB-QX K19-A P162 strain is deposited under accession number CCTCC NO: V202366. The nucleotide sequence of the S1 gene of the IB-QX K19-A P162 strain is described in SEQ ID NO:1; and the amino acid sequence encoded by the S1 gene of the IB-QX K19-A P162 strain (S1 protein) is described in SEQ ID NO:4. Detailed methods for preparing the IB-QX K19-A P162 strain are described in Example 1.

[0034] The term "IB-QX K19-A P171 strain" refers to the 171st generation of the IB-QX K19 strain. The IB-QX K19-A P171 strain is also an attenuated IBV strain, and it is deposited under accession number CCTCC NO: V202367. The nucleotide sequence of the S1 gene of the IB-QX K19-AP171 strain is described in SEQ ID NO:2; and the amino acid sequence encoded by the S1 gene of the IB-QX K19-A P162 strain (S1 protein) is described in SEQ ID NO:5. Detailed methods for preparing the IB-QX K19-A P171 strain are described in Example 1. The term "IB-QX K19-A P171 strain" has a similar meaning to "IB-QX K19-A P162 strain".

[0035] The term "IB-QX K19-A P179 strain" refers to the 179th generation of the IB-QX K19 strain. The IB-QX K19-A P179 strain is also an attenuated IBV strain, and it is deposited under accession number CCTCC NO: V202368. The nucleotide sequence of the S1 gene of the IB-QX K19-AP179 strain is described in SEQ ID NO:3; and the amino acid sequence encoded by the S1 gene of the IB-QX K19-A P162 strain (S1 protein) is described in SEQ ID NO:6. Detailed methods for preparing the IB-QX K19-A P179 strain are described in Example 1. The term "IB-QX K19-A P179 strain" has a similar meaning to "IB-QX K19-A P162 strain".

[0036] In one aspect, the present invention provides an attenuated IBV, wherein the attenuated IBV comprises an S1 gene containing or composed of nucleotide sequences, the nucleotide sequences encoding an amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO:4.

[0037] The term "sequence identity" refers to the relationship between two or more amino acid sequences or two or more nucleic acid sequences (i.e., a reference sequence and a given sequence to be compared with the reference sequence). Sequence identity is determined by comparing a given sequence with a reference sequence, as determined by matching strings of such sequences, after optimal alignment to produce the highest degree of sequence similarity. Methods for calculating sequence identity between two or more amino acid / nucleic acid sequences are well known in the art.

[0038] In some embodiments, the attenuated IBV includes an S1 gene comprising or composed of a nucleotide sequence encoding an amino acid sequence having at least 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO:4. In some embodiments, the attenuated IBV includes an S1 gene comprising or composed of a nucleotide sequence encoding an amino acid sequence having at least 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO:4. In some embodiments, the attenuated IBV includes an S1 gene comprising or composed of nucleotide sequences encoding amino acid sequences selected from the following: SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6.

[0039] In some embodiments, the attenuated IBV includes an S1 gene comprising or composed of a nucleotide sequence having at least 95% sequence identity with the nucleotide sequence shown in SEQ ID NO:1. In some embodiments, the attenuated IBV includes an S1 gene comprising or composed of a nucleotide sequence having at least 96%, 97%, 98%, 99%, or 100% sequence identity with the nucleotide sequence shown in SEQ ID NO:1. In some embodiments, the attenuated IBV includes an S1 gene comprising or composed of a nucleotide sequence having at least 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% sequence identity with the nucleotide sequence shown in SEQ ID NO:1. In some embodiments, the attenuated IBV includes an S1 gene comprising or composed of a nucleotide sequence having at least 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% sequence identity with the nucleotide sequence shown in SEQ ID NO:1. In some embodiments, the S1 gene of the attenuated IBV comprises or is composed of a nucleotide sequence selected from or composed of nucleotide sequences selected from SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3.

[0040] In some implementations, the attenuated IBV has the QX serotype. In some implementations, the attenuated IBV is derived from the IB-QX K19 P7 strain.

[0041] In some embodiments, the attenuated IBV is selected from the following: IB-QX K19-A P162 strain, IB-QX K19-AP171 strain, and IB-QX K19-A P179 strain. In some embodiments, the attenuated IBV is isolated.

[0042] The examples show that the S1 gene / protein of the IB-QX K19-A P162, IB-QX K19-A P171, and IB-QX K19-AP179 strains is almost identical (greater than 99.8% sequence identity, see [link]). Figure 1-6 They are safe for chickens and can protect them from IBV infection.

[0043] In another aspect, the present invention provides an attenuated IBV, wherein the attenuated IBV can be obtained by passage of the IB-QX K19 P7 strain deposited under accession number CCTCC NO: V202364 to at least the 160th generation.

[0044] In some embodiments, attenuated IBV can be obtained by passage the IB-QX K19 P7 strain to generations 160 to 200. In some embodiments, attenuated IBV can be obtained by passage the IB-QX K19 P7 strain to generations 160 to 180. In some embodiments, attenuated IBV can be obtained by passage the IB-QX K19 P7 strain to generations 162 to 179. In some embodiments, attenuated IBV can be obtained by passage the IB-QX K19 P7 strain to the 162nd, 163rd, 164th, 165th, 166th, 167th, 168th, 169th, 170th, 171st, 172nd, 173rd, 174th, 175th, 176th, 177th, 178th, or 179th generation. The term "available" is used herein to define attenuated IBV prepared through its preparation process. For the purposes of this invention, the term "available" encompasses the term "obtained".

[0045] In some embodiments, the attenuated IBV includes an S1 gene comprising or composed of a nucleotide sequence encoding an amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO:4. In some embodiments, the attenuated IBV includes an S1 gene comprising or composed of a nucleotide sequence encoding an amino acid sequence having at least 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO:4. In some embodiments, the attenuated IBV includes an S1 gene comprising or composed of a nucleotide sequence encoding an amino acid sequence having at least 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO:4. In some embodiments, the attenuated IBV includes an S1 gene comprising or composed of a nucleotide sequence encoding an amino acid sequence selected from the following: SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6.

[0046] In some embodiments, the attenuated IBV includes an S1 gene comprising or composed of a nucleotide sequence having at least 95% sequence identity with the nucleotide sequence shown in SEQ ID NO:1. In some embodiments, the attenuated IBV includes an S1 gene comprising or composed of a nucleotide sequence having at least 96%, 97%, 98%, 99%, or 100% sequence identity with the nucleotide sequence shown in SEQ ID NO:1. In some embodiments, the attenuated IBV includes an S1 gene comprising or composed of a nucleotide sequence having at least 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% sequence identity with the nucleotide sequence shown in SEQ ID NO:1. In some embodiments, the attenuated IBV includes an S1 gene comprising or composed of a nucleotide sequence having at least 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% sequence identity with the nucleotide sequence shown in SEQ ID NO:1. In some embodiments, the S1 gene of the attenuated IBV comprises or is composed of a nucleotide sequence selected from or composed of nucleotide sequences selected from SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3.

[0047] In some embodiments, attenuated IBV can be obtained by passage of the IB-QX K19 P7 strain to the 162nd generation, and the S1 gene of the attenuated IBV contains or consists of a nucleotide sequence encoding an amino acid sequence selected from or composed of the following: SEQ ID NO:4, preferably the S1 gene of the attenuated IBV contains or consists of the nucleotide sequence shown in SEQ ID NO:1.

[0048] In some embodiments, attenuated IBV can be obtained by passage of the IB-QX K19 P7 strain to the 171st generation, and the S1 gene of the attenuated IBV contains or consists of a nucleotide sequence encoding an amino acid sequence selected from or composed of the following: SEQ ID NO:5, preferably the S1 gene of the attenuated IBV contains or consists of the nucleotide sequence shown in SEQ ID NO:2.

[0049] In some embodiments, attenuated IBV can be obtained by passage of the IB-QX K19 P7 strain to the 179th generation, and the S1 gene of the attenuated IBV contains or consists of a nucleotide sequence encoding an amino acid sequence selected from or composed of the following: SEQ ID NO:6, preferably the S1 gene of the attenuated IBV contains or consists of the nucleotide sequence shown in SEQ ID NO:3.

[0050] In another aspect, the present invention provides an attenuated infectious bronchitis virus (IBV) selected from the following: IB-QX K19-A P162 strain deposited under accession number CCTCC NO: V202366, IB-QX K19-A P171 strain deposited under accession number CCTCC NO: V202367, and IB-QX K19-A P179 strain deposited under accession number CCTCC NO: V202368.

[0051] In some implementations, the attenuated IBV is isolated. As used herein, the term "isolated" means that the attenuated IBV is not contained within the tissues of the live animal.

[0052] In another aspect, the present invention further provides a method for preparing attenuated IBV, comprising the step of passage an IB-QX K19 P7 strain deposited under accession number CCTCC NO: V202364 into chicken embryos or chicken embryo-derived cells to at least the 160th generation.

[0053] In some embodiments, the method includes the step of passage the IB-QX K19 P7 strain in chicken embryos or chicken embryo-derived cells to the 160th to 200th generation. In some embodiments, the method includes the step of passage the IB-QX K19 P7 strain in chicken embryos or chicken embryo-derived cells to the 160th to 180th generation. In some embodiments, the method includes the step of passage the IB-QX K19 P7 strain in chicken embryos or chicken embryo-derived cells to the 162nd to 179th generation. In some embodiments, the method includes the step of passage the IB-QX K19 P7 strain in chicken embryos or chicken embryo-derived cells to the 162nd, 163rd, 164th, 165th, 166th, 167th, 168th, 169th, 170th, 171st, 172nd, 173rd, 174th, 175th, 176th, 177th, 178th, or 179th generation.

[0054] In some embodiments, the method further includes the step of harvesting allantoic fluid or cell culture from chicken embryos and filtering it to obtain the attenuated IBV strain of the present invention.

[0055] In some embodiments, the chicken embryo-derived cells are primary chicken embryo cells. In some embodiments, the primary chicken embryo cells are fibroblasts or cells derived from kidney, liver, or lung tissue.

[0056] In some embodiments, the attenuated IBV includes an S1 gene comprising or composed of a nucleotide sequence encoding an amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO:4. In some embodiments, the attenuated IBV includes an S1 gene comprising or composed of a nucleotide sequence encoding an amino acid sequence having at least 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO:4. In some embodiments, the attenuated IBV includes an S1 gene comprising or composed of a nucleotide sequence encoding an amino acid sequence having at least 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO:4. In some embodiments, the attenuated IBV includes an S1 gene comprising or composed of a nucleotide sequence encoding an amino acid sequence selected from the following: SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6.

[0057] In some embodiments, the attenuated IBV includes an S1 gene comprising or composed of a nucleotide sequence having at least 95% sequence identity with the nucleotide sequence shown in SEQ ID NO:1. In some embodiments, the attenuated IBV includes an S1 gene comprising or composed of a nucleotide sequence having at least 96%, 97%, 98%, 99%, or 100% sequence identity with the nucleotide sequence shown in SEQ ID NO:1. In some embodiments, the attenuated IBV includes an S1 gene comprising or composed of a nucleotide sequence having at least 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% sequence identity with the nucleotide sequence shown in SEQ ID NO:1. In some embodiments, the attenuated IBV includes an S1 gene comprising or composed of a nucleotide sequence having at least 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% sequence identity with the nucleotide sequence shown in SEQ ID NO:1. In some embodiments, the S1 gene of the attenuated IBV comprises or is composed of a nucleotide sequence selected from or composed of nucleotide sequences selected from SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3.

[0058] In some embodiments, attenuated IBV can be obtained by passage of the IB-QX K19 P7 strain to the 162nd generation, and the S1 gene of the attenuated IBV contains or consists of a nucleotide sequence encoding an amino acid sequence selected from or composed of the following: SEQ ID NO:4, preferably the S1 gene of the attenuated IBV contains or consists of the nucleotide sequence shown in SEQ ID NO:1.

[0059] In some embodiments, attenuated IBV can be obtained by passage of the IB-QX K19 P7 strain to the 171st generation, and the S1 gene of the attenuated IBV contains or consists of a nucleotide sequence encoding an amino acid sequence selected from or composed of the following: SEQ ID NO:5, preferably the S1 gene of the attenuated IBV contains or consists of the nucleotide sequence shown in SEQ ID NO:2.

[0060] In some embodiments, attenuated IBV can be obtained by passage of the IB-QX K19 P7 strain to the 179th generation, and the S1 gene of the attenuated IBV contains or consists of a nucleotide sequence encoding an amino acid sequence selected from or composed of the following: SEQ ID NO:6, preferably the S1 gene of the attenuated IBV contains or consists of the nucleotide sequence shown in SEQ ID NO:3.

[0061] In some embodiments, the attenuated IBV is selected from the following: IB-QX K19-A P162 strain, IB-QX K19-AP171 strain, and IB-QX K19-A P179 strain. In some embodiments, the attenuated IBV is isolated.

[0062] In this invention, the IB-QX K19 strain is used as the parent strain for producing several attenuated IBV strains. The produced attenuated IBV strains are safe for chickens and do not exhibit virulence reversion. Furthermore, the produced attenuated IBV strains demonstrate excellent protective efficacy against IBV infection in chickens, with a protection rate exceeding 90%; among them, the IB-QX K19-A P162 strain shows protection even at levels as low as 10... 3.0 EID 50 Even at chicken doses, it still showed extremely strong protective efficacy.

[0063] Offspring of attenuated IBV This invention also provides any progeny of the attenuated IBV strain preserved in this invention.

[0064] As used in this article, when IBV strain X is passaged starting from IBV strain Y, IBV strain X can be referred to as a descendant of IBV strain Y. For example, the descendant of IB-QX K19-A P162 strain can be obtained by inoculating a certain amount of IB-QXK19-A P162 strain into the allantoic cavity of chicken embryos, incubating the chicken embryos under suitable conditions (e.g., 37°C, 24 hours), harvesting the allantoic fluid from the chicken embryos, and isolating the virus strain. The isolated virus strain is a descendant of IB-QX K19-A P162 strain. The above process can be repeated several times to obtain several descendants of IB-QX K19-A P162 strain at different passages.

[0065] The progeny of the attenuated IBV strain preserved in this invention are also attenuated strains and possess all the identification characteristics of the preserved attenuated IBV strain. "Identification characteristics of the preserved attenuated IBV strain" include, but are not limited to, the S1 gene / protein sequence of the preserved attenuated IBV strain, the protective properties of the preserved attenuated IBV strain against virulent IB-QX challenge, etc.

[0066] In some embodiments, the identifying characteristic of the preserved attenuated IBV strain is the S1 protein sequence of the preserved attenuated IBV strain, or an S1 protein having at least 99%, preferably at least 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% sequence identity with the S1 protein of the preserved attenuated IBV strain. In some embodiments, the identifying characteristic of the preserved attenuated IBV strain is the S1 gene sequence of the preserved attenuated IBV strain, or an S1 gene having at least 99%, preferably at least 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% sequence identity with the S1 gene of the preserved attenuated IBV strain.

[0067] In some embodiments, the identification characteristics of the preserved attenuated IBV strain are protective, providing protection against challenge with virulent IB-QX in at least 80%, preferably 85%, more preferably 90%, even more preferably 95%, and most preferably 100% of poultry. In some embodiments, the identification characteristics of the preserved attenuated IBV strain are protective, providing improved ciliary stagnation scores in at least 90%, preferably 95%, and most preferably 100% of poultry after challenge with virulent IB-QX. In this invention, the term "attenuated IBV" also encompasses any of the progeny of attenuated IBV.

[0068] In some embodiments, the progeny is an attenuated progeny of the IB-QXK19-A P162 strain possessing all the identified characteristics of the IB-QX K19-A P162 strain. In some embodiments, the attenuated progeny of the IB-QX K19-A P162 strain contains the same S1 protein as the IB-QX K19-A P162 strain shown in SEQ ID NO:4, or an S1 protein having at least 99%, preferably at least 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% sequence identity with the S1 protein of the IB-QX K19-A P162 strain shown in SEQ ID NO:4. In some embodiments, the attenuated progeny of the IB-QXK19-A P162 strain contains the same S1 gene (SEQ ID NO:1) as the IB-QX K19-A P162 strain, or has an S1 gene with at least 99%, preferably at least 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% sequence identity with the S1 gene of the IB-QX K19-A P162 strain. In some implementations, attenuated progeny of the IB-QXK19-A P162 strain can protect poultry from attack with the virulent IB-QX, including protection against attack with the virulent IB-QX in at least 80%, preferably 85%, more preferably 90%, more preferably 95%, and most preferably 100% of poultry, or improved ciliary stagnation scores after attack with the virulent IB-QX in at least 90%, preferably 95%, and most preferably 100% of poultry.

[0069] In some embodiments, the progeny is an attenuated progeny of the IB-QXK19-A P171 strain possessing all the identified characteristics of the IB-QX K19-A P171 strain. In some embodiments, the attenuated progeny of the IB-QX K19-A P171 strain contains the same S1 protein as the IB-QX K19-A P171 strain shown in SEQ ID NO:5, or an S1 protein having at least 99%, preferably at least 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% sequence identity with the S1 protein of the IB-QX K19-A P171 strain shown in SEQ ID NO:5. In some embodiments, the attenuated progeny of the IB-QXK19-A P171 strain contains the same S1 gene (SEQ ID NO:2) as the IB-QX K19-A P171 strain, or has an S1 gene with at least 99%, preferably at least 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% sequence identity with the S1 gene of the IB-QX K19-A P171 strain. In some implementations, attenuated progeny of the IB-QXK19-A P171 strain can protect poultry from attack with the virulent IB-QX, including protection against attack with the virulent IB-QX in at least 80%, preferably 85%, more preferably 90%, more preferably 95%, and most preferably 100% of poultry, or improved ciliary stagnation scores after attack with the virulent IB-QX in at least 90%, preferably 95%, and most preferably 100% of poultry.

[0070] In some embodiments, the progeny is an attenuated progeny of the IB-QXK19-A P179 strain possessing all the identified characteristics of the IB-QX K19-A P179 strain. In some embodiments, the attenuated progeny of the IB-QX K19-A P179 strain contains the same S1 protein as the IB-QX K19-A P179 strain shown in SEQ ID NO:6, or an S1 protein having at least 99%, preferably at least 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% sequence identity with the S1 protein of the IB-QX K19-A P179 strain shown in SEQ ID NO:6. In some embodiments, the attenuated progeny of the IB-QXK19-A P179 strain contains the same S1 gene (SEQ ID NO:3) as the IB-QX K19-A P179 strain, or has an S1 gene with at least 99%, preferably at least 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% sequence identity with the S1 gene of the IB-QX K19-A P179 strain. In some implementations, attenuated progeny of the IB-QXK19-A P179 strain can protect poultry from attack with the virulent IB-QX, including protection against attack with the virulent IB-QX in at least 80%, preferably 85%, more preferably 90%, more preferably 95%, and most preferably 100% of poultry, or improved ciliary stagnation scores after attack with the virulent IB-QX in at least 90%, preferably 95%, and most preferably 100% of poultry.

[0071] In some embodiments, the progeny comprises up to 10 generations of the attenuated IBV strain preserved in this invention. In some embodiments, the progeny comprises up to 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 generations of the attenuated IBV strain preserved in this invention.

[0072] In some embodiments, the offspring contain up to 10 generations of the IB-QX K19-A P162 strain. In some embodiments, the offspring contain up to 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 generations of the IB-QX K19-A P162 strain. In some embodiments, the offspring are the 10th, 9th, 8th, 7th, 6th, 5th, 4th, 3rd, 2nd, or 1st generation of the IB-QX K19-A P162 strain. In some embodiments, the offspring are the 5th, 4th, 3rd, 2nd, or 1st generation of the IB-QX K19-A P162 strain. In some embodiments, the offspring can be obtained by subculturing the IB-QX K19-A P162 strain for 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 generation. In some implementations, offspring can be obtained by passage the IB-QX K19-A P162 strain for 5, 4, 3, 2 or 1 generations.

[0073] In some embodiments, the offspring contain up to 10 generations of the IB-QX K19-A P171 strain. In some embodiments, the offspring contain up to 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 generations of the IB-QX K19-A P171 strain. In some embodiments, the offspring are the 10th, 9th, 8th, 7th, 6th, 5th, 4th, 3rd, 2nd, or 1st generation of the IB-QX K19-A P171 strain. In some embodiments, the offspring are the 5th, 4th, 3rd, 2nd, or 1st generation of the IB-QX K19-A P171 strain. In some embodiments, the offspring can be obtained by subculturing the IB-QX K19-A P171 strain for 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 generation. In some implementations, offspring can be obtained by passage the IB-QX K19-A P171 strain for 5, 4, 3, 2 or 1 generations.

[0074] In some embodiments, the offspring contain up to 10 generations of the IB-QX K19-A P179 strain. In some embodiments, the offspring contain up to 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 generations of the IB-QX K19-A P179 strain. In some embodiments, the offspring are the 10th, 9th, 8th, 7th, 6th, 5th, 4th, 3rd, 2nd, or 1st generation of the IB-QX K19-A P179 strain. In some embodiments, the offspring are the 5th, 4th, 3rd, 2nd, or 1st generation of the IB-QX K19-A P179 strain. In some embodiments, the offspring can be obtained by subculturing the IB-QX K19-A P179 strain for 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 generation. In some implementations, offspring can be obtained by passage the IB-QX K19-A P179 strain for 5, 4, 3, 2 or 1 generations.

[0075] In another aspect, the present invention provides an attenuated infectious bronchitis virus (IBV) selected from: the IB-QX K19-A P162 strain deposited under accession number CCTCC NO: V202366, any of the progeny of the IB-QX K19-A P162 strain comprising up to 5, 4, 3, 2, or 1 generations (e.g., the 5th, 4th, 3rd, 2nd, or 1st generation of the IB-QX K19-A P162 strain), the IB-QX K19-A P171 strain deposited under accession number CCTCC NO: V202367, any of the progeny of the IB-QX K19-A P171 strain comprising up to 5, 4, 3, 2, or 1 generations (e.g., the IB-QX K19-A P171 strain). The 5th, 4th, 3rd, 2nd or 1st generation of the K19-A P171 strain, the IB-QX K19-A P179 strain deposited under accession number CCTCC NO: V202368, and any of the progeny of the IB-QX K19-A P179 strain containing the IB-QX K19-A P179 strain at most 5th, 4th, 3rd, 2nd or 1st generation (e.g., the 5th, 4th, 3rd, 2nd or 1st generation of the IB-QX K19-A P179 strain).

[0076] In another aspect, the present invention provides a method for preparing any progeny of the preserved attenuated IBV strain of the present invention.

[0077] In some embodiments, the method includes the step of passage the IB-QXK19-A P162 strain deposited under accession number CCTCC NO: V202366 up to 10 times in chicken embryos or chicken embryo-derived cells. In some embodiments, the method includes the step of passage the IB-QX K19-A P162 strain deposited under accession number CCTCC NO: V202366 into chicken embryos or chicken embryo-derived cells for 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 times. In some embodiments, the method includes the step of passage the IB-QX K19-A P162 strain deposited under accession number CCTCC NO: V202366 into chicken embryos or chicken embryo-derived cells for 5, 4, 3, 2, or 1 times.

[0078] In some embodiments, the method includes the step of passage the IB-QXK19-A P171 strain deposited under accession number CCTCC NO: V202367 in chicken embryos or chicken embryo-derived cells for up to 10 generations. In some embodiments, the method includes the step of passage the IB-QX K19-A P171 strain deposited under accession number CCTCC NO: V202367 in chicken embryos or chicken embryo-derived cells for 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 generation. In some embodiments, the method includes the step of passage the IB-QX K19-A P171 strain deposited under accession number CCTCC NO: V202367 in chicken embryos or chicken embryo-derived cells for 5, 4, 3, 2, or 1 generation.

[0079] In some embodiments, the method includes the step of passage the IB-QXK19-A P179 strain deposited under accession number CCTCC NO: V202368 up to 10 generations in chicken embryos or chicken embryo-derived cells. In some embodiments, the method includes the step of passage the IB-QX K19-A P179 strain deposited under accession number CCTCC NO: V202368 for 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 generations in chicken embryos or chicken embryo-derived cells. In some embodiments, the method includes the step of passage the IB-QX K19-A P179 strain deposited under accession number CCTCC NO: V202368 for 5, 4, 3, 2, or 1 generations in chicken embryos or chicken embryo-derived cells.

[0080] In some embodiments, the method further includes the step of harvesting allantoic fluid or cell culture from chicken embryos and filtering it to obtain progeny of the preserved attenuated IBV strain of the present invention.

[0081] In some embodiments, the chicken embryo-derived cells are primary chicken embryo cells. In some embodiments, the primary chicken embryo cells are fibroblasts or cells derived from kidney, liver, or lung tissue. In some embodiments, the attenuated IBV is isolated.

[0082] Surprisingly, in the case studies, it was found that the progeny of the preserved attenuated IBV strain retained the same immunological properties as the preserved attenuated IBV strain, and the preserved attenuated IBV strain and its progeny within at least 10 generations showed excellent immunoprotective efficacy against virulent IB-QX challenge in chickens.

[0083] Immunogenic compositions and vaccines This invention provides an immunogenic composition or a live attenuated vaccine against IBV infection.

[0084] 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. As used herein, an immunogenic composition confers protective immunity against one or more clinical signs of IBV infection. An immunogenic composition is described as a "vaccine" where the host exhibits a protective immune response that enhances resistance to new infections and / or reduces the clinical severity of the disease.

[0085] In one aspect, the present invention provides an immunogenic composition or a live attenuated vaccine against IBV infection.

[0086] In some embodiments, the immunogenic composition or attenuated live vaccine comprises a therapeutically or preventively effective amount of the attenuated IBV of the present invention, or a therapeutically or preventively effective amount of attenuated IBV prepared by the method of the present invention.

[0087] The term "effective amount" refers to the amount of antigen that elicits or is capable of eliciting an immune response in poultry. Such effective amounts can reduce the incidence of a specific IBV infection or reduce the severity of the clinical signs of a specific IBV infection in poultry. In some embodiments, compared with poultry not treated / prevented with the attenuated IBV, immunogenic composition, or vaccine of the present invention, the clinical signs are reduced in morbidity or severity by at least 10%, more preferably at least 20%, even more preferably at least 30%, even more preferably at least 40%, even more preferably at least 50%, even more preferably at least 60%, even more preferably at least 70%, even more preferably at least 80%, even more preferably at least 90%, even more preferably at least 95%, and most preferably 100%.

[0088] In some embodiments, the immunogenic composition or live attenuated vaccine contains 1 to 10 log [unclear text - possibly a number of ingredients]. 10 EID 50The attenuated IBV doses per ml include 1, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, and 8.0 log. 10 EID 50 / ml of attenuated IBV. In some embodiments, the immunogenic composition or attenuated live vaccine contains 2 to 8 log [amount missing]. 10 EID 50 / ml of attenuated IBV. In some embodiments, the immunogenic composition or attenuated live vaccine contains 3 to 8 log [amount missing]. 10 EID 50 / ml of attenuated IBV. In some embodiments, the immunogenic composition or attenuated live vaccine contains 5 to 8 log [units unspecified]. 10 EID 50 / ml of attenuated IBV. In some embodiments, the immunogenic composition or attenuated live vaccine contains 6 to 7 log [units of measurement missing]. 10 EID 50 / ml of attenuated IBV. In some embodiments, the immunogenic composition or attenuated live vaccine contains 6.5 log 10 EID 50 / ml is the amount of IBV that is reduced.

[0089] In some embodiments, the immunogenic composition or attenuated live vaccine comprises at least one veterinary-acceptable carrier. In some embodiments, the veterinary-acceptable carrier is a lyophilized protectant.

[0090] In some embodiments, the immunogenic composition or live attenuated vaccine is formulated for single-dose administration. In some embodiments, the immunogenic composition or live attenuated vaccine is administered via eye drops, nasal drops, or ocular / nasal drops. In some embodiments, the immunogenic composition or live attenuated vaccine is administered via a spray.

[0091] In another aspect, the present invention provides a method for preparing an immunogenic composition or a live attenuated vaccine against infectious bronchitis virus (IBV) infection.

[0092] In some implementations, the method includes the following steps: (1) Preparation of the attenuated IBV of the present invention; (2) Add a veterinary-acceptable carrier, such as a lyophilized protectant, and mix to obtain an immunogenic composition or a live attenuated vaccine against IBV infection.

[0093] The attenuated IBV of the present invention has been described above. An exemplary method for preparing the attenuated IBV of the present invention has also been described above.

[0094] In some embodiments, the method for preparing the attenuated IBV of the present invention includes the following steps: passage the IB-QX K19 P7 strain, deposited under accession number CCTCC NO: V202364, in chicken embryos or chicken embryo-derived cells to at least the 160th generation; preferably, passage the IB-QX K19 P7 strain in chicken embryos or chicken embryo-derived cells to the 160th to 200th generation; more preferably, passage the IB-QX K19 P7 strain in chicken embryos or chicken embryo-derived cells to the 160th to 180th generation; even more preferably, passage the IB-QX K19 P7 strain in chicken embryos or chicken embryo-derived cells to the 162nd to 179th generation; even more preferably, passage the IB-QX K19 P7 strain in chicken embryos or chicken embryo-derived cells to the 162nd to 179th generation; even more preferably, passage the IB-QX K19 P7 strain in chicken embryos or chicken embryo-derived cells to the 160th to 179th generation. The P7 strain was passaged in chicken embryos or chicken embryo-derived cells to the 162nd, 163rd, 164th, 165th, 166th, 167th, 168th, 169th, 170th, 171st, 172nd, 173rd, 174th, 175th, 176th, 177th, 178th, or 179th generation; most preferably, the IB-QX K19 P7 strain was passaged in chicken embryos or chicken embryo-derived cells to the 162nd, 171st, or 179th generation. The method for preparing attenuated IBV of the present invention is the same as described above.

[0095] The attenuated IBV strains of the present invention exhibit excellent immunogenicity over a wide range of generations up to 20, and are therefore well-suited for the industrial production of immunogenic compositions or attenuated live IBV vaccines.

[0096] Treatment and prevention methods This invention provides a method for immunizing poultry, or a method for treating or preventing clinical signs caused by IBV in poultry, or a method for reducing ciliary stagnation in poultry.

[0097] In one aspect, the present invention provides a method for immunizing poultry, comprising the step of administering to such poultry a therapeutically or preventively effective amount of the attenuated IBV, immunogenic composition, or vaccine of the present invention.

[0098] In one aspect, the present invention provides an immunogenic composition or vaccine of the present invention for immunizing poultry, the method comprising the step of administering to such poultry a therapeutically or preventively effective amount of the attenuated IBV, immunogenic composition or vaccine of the present invention.

[0099] In one aspect, the present invention provides the use of the attenuated IBV of the present invention in the preparation of the immunogenic composition or vaccine of the present invention for immunizing poultry.

[0100] The term "immunization" refers to active immunization, by administering the attenuated IBV, immunogenic composition, or vaccine of the present invention to poultry to be immunized, thereby inducing an immune response against antigens included in such attenuated IBV, immunogenic composition, or vaccine of the present invention. Immunization results in a reduction in the incidence of a specific IBV infection in poultry, or a reduction in the severity of clinical signs caused by or associated with a specific IBV infection.

[0101] It should be understood that immunization may not be effective in every immunized individual of poultry. However, the term requires that a significant portion of the poultry be effectively immunized. In some embodiments, immunization is considered effective if, compared to poultry not immunized with the attenuated IBV, immunogenic composition, or vaccine of the present invention, 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 poultry to be immunized show a reduction in morbidity or severity of clinical signs by at least 50%, more preferably at least 60%, more preferably at least 70%, even more preferably at least 80%, more preferably at least 90%, more preferably at least 95%, and most preferably 100%.

[0102] The term "poultry" is well known to those skilled in the art. Throughout this invention, the term "poultry" encompasses domestic fowl, including, for example, chickens, turkeys, quails, pheasants, guinea fowl, geese, and ducks. The term "chicken" includes broilers, laying hens, and breeding stock (also referred to as breeder chickens) for both. In some embodiments, poultry refers to domestic fowl. In some embodiments, domestic fowl is selected from chickens, turkeys, quails, pheasants, guinea fowl, geese, and ducks. In some embodiments, domestic fowl is selected from chickens, turkeys, quails, and pheasants. In some embodiments, chickens are selected from broilers, laying hens, and breeder chickens.

[0103] In some implementations, the poultry to be immunized are within the previous week, within the previous three days, within the previous two days, or within the previous day. In some implementations, the poultry to be immunized are 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 days old. In some implementations, the poultry to be immunized are 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days old. In some implementations, the poultry to be immunized are 1, 2, 3, 4, 5, 6, or 7 days old. In some implementations, the poultry to be immunized are within the previous day. In some implementations, the poultry are chickens, and the chickens are within the previous day.

[0104] In one aspect, the present invention provides a method for treating or preventing clinical signs caused by IBV in poultry, comprising the step of administering to poultry a therapeutically or preventively effective amount of the attenuated IBV, immunogenic composition, or vaccine of the present invention.

[0105] In one aspect, the present invention provides an immunogenic composition or vaccine of the present invention for a method of treating or preventing clinical signs caused by IBV in poultry, the method comprising the step of administering to poultry a therapeutically or preventively effective amount of attenuated IBV, immunogenic composition or vaccine of the present invention.

[0106] In one aspect, the present invention provides the use of the attenuated IBV of the present invention in the preparation of an immunogenic composition or vaccine of the present invention for the treatment or prevention of clinical signs caused by IBV in poultry.

[0107] The term "treating" refers to the administration of an effective amount of the attenuated IBV, immunogenic composition, or vaccine of the present invention once poultry has been infected with this type of IBV and / or has shown some clinical signs caused or associated with such IBV infection. The term "preventing" refers to administration to poultry prior to any IBV infection, or at least before such poultry has shown any clinical signs caused or associated with infection by this type of IBV.

[0108] The term "clinical signs" refers to the signs of IBV infection in poultry. In some implementation plans, clinical signs caused by IBV infection are selected from the following: ciliary arrest, rales, decreased egg production, kidney damage, watery diarrhea, weight loss, viral load, viral shedding, and combinations thereof. In some implementation plans, the clinical sign caused by IBV is ciliary arrest.

[0109] In one aspect, the present invention provides a method for reducing ciliary stagnation in poultry compared to non-immunized poultry, comprising the step of administering to poultry a therapeutically or preventively effective amount of the attenuated IBV, immunogenic composition, or vaccine of the present invention.

[0110] In one aspect, the present invention provides an immunogenic composition or vaccine of the present invention for a method of reducing ciliary stagnation in poultry compared to unimmunized poultry, the method comprising the step of administering to poultry a therapeutically or preventively effective amount of the attenuated IBV, immunogenic composition or vaccine of the present invention.

[0111] In one aspect, the present invention provides the use of the attenuated IBV of the present invention in the preparation of the immunogenic composition or vaccine of the present invention, wherein the immunogenic composition or vaccine reduces ciliary stagnation in poultry compared to unimmunized poultry.

[0112] The term "ciliary arrest" is well known to those skilled in the art. The trachea is lined with specialized epithelial cells, which are lined with numerous, active, hair-like structures called cilia. The term "ciliary arrest" encompasses the reduction or loss of cilia and / or the loss or partial loss of ciliary activity (motility). Ciliary arrest can be determined by examining the lining of the tracheal rings in relation to ciliary movement. How to determine ciliary movement in the trachea is within the general knowledge of those skilled in the art.

[0113] The term "reduction in ciliary stasis / reduction in ciliary stasis" means that, compared with non-immunized poultry, ciliary stasis is reduced by at least 10%, preferably at least 20%, more preferably at least 30%, even more preferably at least 40%, even more preferably at least 50%, even more preferably at least 60%, even more preferably at least 70%, even more preferably at least 80%, even more preferably at least 90%, even more preferably at least 95%, and most preferably 100%. How to measure the reduction in ciliary stasis is within the general knowledge of those skilled in the art, and exemplary methods for evaluating the reduction in ciliary stasis are described in Example 3.

[0114] In some embodiments, the immunogenic compositions or vaccines of the present invention are formulated for single-dose administration.

[0115] In some embodiments, the immunogenic composition or vaccine of the present invention is administered via eye drops, nasal drops, or ocular / nasal drops. In some embodiments, the immunogenic composition or vaccine of the present invention is administered via a spray.

[0116] In some embodiments, the immunogenic composition or vaccine of the present invention contains 1 to 10 log... 10 EID 50 The attenuated IBV doses per ml include 1, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, and 8.0 log. 10 EID 50 / ml attenuated IBV amount. In some embodiments, the immunogenic composition or vaccine of the present invention contains 2 to 8 log 10 EID 50 / ml of attenuated IBV. In some embodiments, the immunogenic composition or attenuated live vaccine of the present invention contains 3 to 8 logs. 10 EID 50 / ml attenuated IBV amount. In some embodiments, the immunogenic composition or vaccine of the present invention contains 5 to 8 log 10 EID 50 / ml attenuated IBV amount. In some embodiments, the immunogenic composition or vaccine of the present invention contains 6 to 7 log 10EID 50 / ml attenuated IBV amount. In some embodiments, the immunogenic composition or vaccine of the present invention contains 6.5 log 10 EID 50 / ml is the amount of IBV that is reduced.

[0117] The examples show that a single dose of the attenuated IBV of the present invention can provide sufficient protection against IBV infection in 1-day-old chickens via eye or nasal drops or spray.

[0118] Reagent test kit When necessary, the immunogenic composition or vaccine of the present invention may be contained in a packaging or dispenser device, which may contain one or more unit dosage forms containing an active antigenic component. The packaging may, for example, comprise metal or plastic foil, such as blister packs. The packaging or dispenser device may be accompanied by instructions for use, preferably for poultry, especially birds. Associated with such containers may be a notification in the form prescribed by a government agency regulating the manufacture, use, or sale of the drug or biological product, reflecting approval for administration by the manufacturing, using, or selling agency.

[0119] In one aspect, the present invention provides a kit comprising the attenuated IBV, immunogenic composition, or vaccine of the present invention. In some embodiments, the kit further comprises instructions for treating and / or preventing IBV infection in poultry. In some embodiments, the kit further comprises a dispenser capable of administering the attenuated IBV, immunogenic composition, or vaccine of the present invention to poultry.

[0120] The following technical solutions are also described herein and constitute part of the disclosure of this invention: Technical solution Technical Solution Set A 1A. An attenuated infectious bronchitis virus (IBV), wherein the attenuated IBV comprises an S1 gene containing or composed of nucleotide sequences, the nucleotide sequences encoding an amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO:4; Preferably, the attenuated IBV comprises an S1 gene containing or composed of nucleotide sequences, the nucleotide sequences encoding an amino acid sequence having at least 96%, 97%, 98%, 99% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO:4; More preferably, the attenuated IBV comprises an S1 gene containing or composed of nucleotide sequences, the nucleotide sequences encoding an amino acid sequence having at least 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO:4; or The attenuated IBV includes an S1 gene comprising or composed of nucleotide sequences, the nucleotide sequences encoding amino acid sequences selected from the following: SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6.

[0121] 2A. The attenuated IBV of technical solution 1A, wherein the S1 gene comprises or is composed of a nucleotide sequence, the nucleotide sequence having at least 95% sequence identity with the nucleotide sequence shown in SEQ ID NO:1; Preferably, the S1 gene comprises or is composed of nucleotide sequences, wherein the nucleotide sequences have at least 96%, 97%, 98%, 99% or 100% sequence identity with the nucleotide sequence shown in SEQ ID NO:1; More preferably, the S1 gene comprises or is composed of a nucleotide sequence, the nucleotide sequence having at least 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% sequence identity with the nucleotide sequence shown in SEQ ID NO:1; or The S1 gene comprises or consists of nucleotide sequences selected from or composed of nucleotide sequences selected from the following: SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3.

[0122] 3A. The attenuated IBV of technical solution 1A or 2A, wherein the attenuated IBV has the QX serotype; preferably, wherein the attenuated IBV is derived from the IB-QX K19 P7 strain deposited under accession number CCTCCNO: V202364.

[0123] 4A. The attenuated IBV of any one of technical solutions 1A to 3A, wherein the attenuated IBV is at least the 160th generation; preferably, the 160th to 200th generation; more preferably, the 160th to 180th generation; even more preferably, the 162nd to 179th generation; even more preferably, the 162nd, 163rd, 164th, 165th, 166th, 167th, 168th, 169th, 170th, 171st, 172nd, 173rd, 174th, 175th, 176th, 177th, 178th or 179th generation; and most preferably, the 162nd, 171st or 179th generation.

[0124] 5A. An attenuated infectious bronchitis virus (IBV), wherein the attenuated IBV can be obtained by passage of the IB-QX K19 P7 strain deposited under accession number CCTCC NO: V202364 to at least 160 generations; preferably 160 to 200 generations; more preferably 160 to 180 generations; more preferably 162 to 179 generations; more preferably 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178 or 179 generations; and most preferably 162, 171 or 179 generations.

[0125] 6A. The attenuated IBV of technical solution 5A, wherein the attenuated IBV comprises an S1 gene containing or composed of nucleotide sequences, the nucleotide sequences encoding an amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO:4; Preferably, the attenuated IBV comprises an S1 gene containing or composed of nucleotide sequences, the nucleotide sequences encoding an amino acid sequence having at least 96%, 97%, 98%, 99% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO:4; More preferably, the attenuated IBV comprises an S1 gene containing or composed of nucleotide sequences, the nucleotide sequences encoding an amino acid sequence having at least 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO:4; or The attenuated IBV includes an S1 gene comprising or composed of nucleotide sequences, the nucleotide sequences encoding amino acid sequences selected from the following: SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6.

[0126] 7A. Attenuated IBV of technical solution 5A or 6A, wherein the S1 gene comprises or is composed of a nucleotide sequence, the nucleotide sequence having at least 95% sequence identity with the nucleotide sequence shown in SEQ ID NO:1; Preferably, the S1 gene comprises or is composed of nucleotide sequences, wherein the nucleotide sequences have at least 96%, 97%, 98%, 99% or 100% sequence identity with the nucleotide sequence shown in SEQ ID NO:1; More preferably, the S1 gene comprises or is composed of a nucleotide sequence, the nucleotide sequence having at least 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% sequence identity with the nucleotide sequence shown in SEQ ID NO:1; or The S1 gene comprises or consists of nucleotide sequences selected from or composed of nucleotide sequences selected from the following: SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3.

[0127] 8A. An attenuated infectious bronchitis virus (IBV). The attenuated IBV mentioned therein refers to the IB-QX K19-A P162 strain and its progeny deposited under accession number CCTCC NO: V202366, and the progeny of the IB-QX K19-A P162 strain is any attenuated progeny IBV possessing all the identifying characteristics of the deposited IB-QX K19-A P162 strain, including, for example, the same S1 protein as the IB-QX K19-A P162 strain shown in SEQ ID NO:4, or the same S1 protein as the IB-QX K19-A P162 strain shown in SEQ ID NO:4. The S1 protein of strain P162 has at least 99%, preferably at least 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% sequence identity, and / or protection against virulent IB-QX attack, including protection against virulent IB-QX attack in at least 80%, preferably 85%, more preferably 90%, more preferably 95% and most preferably 100% of poultry, or improved ciliary stagnation score after virulent IB-QX attack in at least 90%, preferably 95% and most preferably 100% of poultry; The attenuated IBV mentioned therein refers to the IB-QX K19-A P171 strain and its progeny deposited under accession number CCTCC NO: V202367, and the progeny of the IB-QX K19-A P171 strain is any attenuated progeny IBV possessing all the identifying characteristics of the deposited IB-QX K19-A P171 strain, including, for example, the same S1 protein as the IB-QX K19-A P171 strain shown in SEQ ID NO:5, or the same S1 protein as the IB-QX K19-A P171 strain shown in SEQ ID NO:5. The S1 protein of strain P171 has at least 99%, preferably at least 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% sequence identity, and / or protection against virulent IB-QX attack, including protection against virulent IB-QX attack in at least 80%, preferably 85%, more preferably 90%, more preferably 95%, and most preferably 100% of poultry, or improved ciliary stagnation score after virulent IB-QX attack in at least 90%, preferably 95%, and most preferably 100% of poultry; or The attenuated IBV mentioned therein refers to the IB-QX K19-A P179 strain and its progeny deposited under accession number CCTCC NO: V202368, and the progeny of the IB-QX K19-A P179 strain is any attenuated progeny IBV possessing all the identifying characteristics of the deposited IB-QX K19-A P179 strain, including, for example, the same S1 protein as the IB-QX K19-A P179 strain shown in SEQ ID NO: 6, or the same S1 protein as the IB-QX K19-A P179 strain shown in SEQ ID NO: 6. The S1 protein of strain P179 has at least 99%, preferably at least 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% sequence identity, and / or protection against virulent IB-QX attack, including protection against virulent IB-QX attack in at least 80%, preferably 85%, more preferably 90%, more preferably 95% and most preferably 100% of poultry, or improved ciliary stagnation score after virulent IB-QX attack in at least 90%, preferably 95% and most preferably 100% of poultry.

[0128] 9A. Technical solution 8A's attenuated IBV, in which... - The offspring of the IB-QX K19-A P162 strain have the same S1 gene as the IB-QX K19-A P162 strain shown in SEQ ID NO:1, or have an S1 gene with at least 99%, preferably at least 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% sequence identity with the S1 gene of the IB-QX K19-A P162 strain shown in SEQ ID NO:1; - The offspring of the IB-QX K19-A P171 strain possess the same S1 gene as the IB-QX K19-A P162 strain shown in SEQ ID NO:2, or have an S1 gene with at least 99%, preferably at least 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% sequence identity with the S1 gene of the IB-QX K19-A P162 strain shown in SEQ ID NO:2; or - The offspring of the IB-QX K19-A P179 strain have the same S1 gene as the IB-QX K19-A P162 strain shown in SEQ ID NO:3, or have an S1 gene with at least 99%, preferably at least 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% sequence identity with the S1 gene of the IB-QX K19-A P162 strain shown in SEQ ID NO:3.

[0129] 10A. Technical solution 8A or 9A for attenuated IBV, among which... - The offspring of the IB-QX K19-A P162 strain include at most 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 generations of the IB-QX K19-A P162 strain; or the offspring of the IB-QX K19-A P162 strain are the 10th, 9th, 8th, 7th, 6th, 5th, 4th, 3rd, 2nd, or 1st generation of the IB-QX K19-A P162 strain; or the offspring of the IB-QX K19-A P162 strain can be obtained by subculturing the IB-QX K19-A P162 strain for 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 generation; preferably, the offspring of the IB-QX K19-A P162 strain can be obtained by subculturing the IB-QX K19-A P162 strain for at most 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 generation of the IB-QX K19-A P162 strain. P162 strain is obtained by passing it through 5, 4, 3, 2 or 1 generations; - The offspring of the IB-QX K19-A P171 strain include at most 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 generations of the IB-QX K19-A P171 strain; or the offspring of the IB-QX K19-A P171 strain are the 10th, 9th, 8th, 7th, 6th, 5th, 4th, 3rd, 2nd, or 1st generation of the IB-QX K19-A P171 strain; or the offspring of the IB-QX K19-A P171 strain can be obtained by subculturing the IB-QX K19-A P171 strain for 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 generation; preferably, the offspring of the IB-QX K19-A P171 strain can be obtained by subculturing the IB-QX K19-A P171 strain for at most 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 generation of the IB-QX K19-A P171 strain. P171 strain was obtained by passage 5, 4, 3, 2 or 1 times; or - The offspring of the IB-QX K19-A P179 strain include at most 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 generations of the IB-QX K19-A P179 strain; or the offspring of the IB-QX K19-A P179 strain are the 10th, 9th, 8th, 7th, 6th, 5th, 4th, 3rd, 2nd, or 1st generation of the IB-QX K19-A P179 strain; or the offspring of the IB-QX K19-A P179 strain can be obtained by subculturing the IB-QX K19-A P179 strain for 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 generation; preferably, the offspring of the IB-QX K19-A P179 strain can be obtained by subculturing the IB-QX K19-A P179 strain for at most 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 generation of the IB-QX K19-A P179 strain. P179 strain is obtained by passing it through 5, 4, 3, 2 or 1 generations.

[0130] 11A. An attenuated infectious bronchitis virus (IBV), wherein the attenuated IBV is selected from: the IB-QX K19-A P162 strain deposited under accession number CCTCC NO: V202366, any of the progeny of the IB-QX K19-A P162 strain comprising up to 5, 4, 3, 2, or 1 generations, the IB-QX K19-A P171 strain deposited under accession number CCTCC NO: V202367, any of the progeny of the IB-QX K19-A P171 strain comprising up to 5, 4, 3, 2, or 1 generations, the IB-QX K19-A P179 strain deposited under accession number CCTCC NO: V202368, and the IB-QX K19-A P179 strain comprising IB-QX K19-A P171. Any of the offspring of the IB-QXK19-A P179 strain at most 5, 4, 3, 2 or 1 generations.

[0131] 12A. The attenuated IBV of any one of technical solutions 1A-11A, wherein the attenuated IBV is separate.

[0132] Technical Solution Set B 1B. A method for preparing attenuated infectious bronchitis virus (IBV), comprising passage an IB-QX K19 P7 strain deposited under accession number CCTCC NO: V202364 in chicken embryos or chicken embryo-derived cells to at least 160 generations, preferably 160 to 200 generations, more preferably 160 to 180 generations, more preferably 162 to 179 generations, more preferably 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178 or 179 generations, and most preferably 162, 171 or 179 generations.

[0133] 2B. The method of technical solution 1B, wherein the chicken embryo-derived cells are primary chicken embryo cells; more preferably, the primary chicken embryo cells are fibroblasts or cells derived from kidney, liver or lung tissue.

[0134] 3B. The method of technical solution 1B or 2B, wherein the attenuated IBV comprises an S1 gene containing or composed of nucleotide sequences, the nucleotide sequences encoding an amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO:4; Preferably, the attenuated IBV comprises an S1 gene containing or composed of nucleotide sequences, the nucleotide sequences encoding an amino acid sequence having at least 96%, 97%, 98%, 99% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO:4; More preferably, the attenuated IBV comprises an S1 gene containing or composed of nucleotide sequences, the nucleotide sequences encoding an amino acid sequence having at least 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO:4; or The attenuated IBV includes an S1 gene comprising or composed of nucleotide sequences, the nucleotide sequences encoding amino acid sequences selected from the following: SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6.

[0135] 4B. A method according to any one of technical solutions 1B to 3B, wherein the attenuated IBV comprises an S1 gene containing or composed of nucleotide sequences, said nucleotide sequence having at least 95% sequence identity with the nucleotide sequence shown in SEQ ID NO:1, preferably having at least 96%, 97%, 98%, 99%, or 100% sequence identity with the nucleotide sequence shown in SEQ ID NO:1, more preferably having at least 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% sequence identity with the nucleotide sequence shown in SEQ ID NO:1, more preferably having at least 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% sequence identity with the nucleotide sequence shown in SEQ ID NO:1, more preferably having at least 98.1%, 98.2%, 98.3%, 99.4%, 9 ... The nucleotide sequence shown in NO:1 has at least 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% sequence identity, and most preferably has at least 99.9% or 100% sequence identity with the nucleotide sequence shown in SEQ ID NO:1; or The attenuated IBV includes an S1 gene comprising or composed of nucleotide sequences selected from the following: SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3.

[0136] 5B. A method of any one of technical solutions 1B to 4B, wherein the attenuated IBV can be obtained by passage of the IB-QX K19P7 strain to the 162nd generation, and the S1 gene of the attenuated IBV comprises or consists of a nucleotide sequence encoding an amino acid sequence selected from or composed of the following: SEQ ID NO:4, preferably the S1 gene of the attenuated IBV comprises or consists of the nucleotide sequence shown in SEQ ID NO:1; or The attenuated IBV is obtained by passage of the IB-QX K19 P7 strain to the 171st generation, and the S1 gene of the attenuated IBV contains or is composed of a nucleotide sequence encoding an amino acid sequence selected from or consisting of the following: SEQ ID NO:5, preferably the S1 gene of the attenuated IBV contains or is composed of the nucleotide sequence shown in SEQ ID NO:2; or The attenuated IBV is obtained by passage of the IB-QX K19 P7 strain to the 179th generation, and the S1 gene of the attenuated IBV contains or is composed of a nucleotide sequence encoding an amino acid sequence selected from or composed of the following: SEQ ID NO:6, and the S1 gene of the attenuated IBV contains or is composed of the nucleotide sequence shown in SEQ ID NO:3.

[0137] 6B. The method of any one of technical solutions 1B to 5B, wherein the attenuated IBV is selected from the following: IB-QX K19-A P162 strain deposited under accession number CCTCC NO: V202366, IB-QX K19-A P171 strain deposited under accession number CCTCC NO: V202367, and IB-QX K19-AP179 strain deposited under accession number CCTCC NO: V202368.

[0138] 7B. A method for preparing attenuated infectious bronchitis virus (IBV), comprising the following steps: The IBV strain is passaged in chicken embryos or chicken embryo-derived cells for no more than 10 generations; more preferably, the IBV strain is passaged in chicken embryos or chicken embryo-derived cells for 5, 4, 3, 2 or 1 generations, wherein the IBV strain is selected from the IB-QX K19-A P162 strain deposited under accession number CCTCC NO: V202366, the IB-QXK19-A P171 strain deposited under accession number CCTCC NO: V202367, and the IB-QX K19-A P179 strain deposited under accession number CCTCC NO: V202368.

[0139] 8B. The method of technical solution 7B, wherein the chicken embryo-derived cells are primary chicken embryo cells; more preferably, the primary chicken embryo cells are fibroblasts or cells derived from kidney, liver or lung tissue.

[0140] 9B. The method of any one of technical solutions 1B to 8B, wherein the attenuated IBV is isolated.

[0141] Technical solution set C C1. An immunogenic composition or live attenuated vaccine against infectious bronchitis virus (IBV) infection, comprising a therapeutically or preventively effective amount of attenuated IBV from any of the techniques in set A, or a therapeutically or preventively effective amount of attenuated IBV prepared by any of the techniques in set B. Preferably, the amount of attenuated IBV is 1 to 10 log. 10 EID 50 / ml attenuated IBV; more preferably, the amount of attenuated IBV is 2 to 8 log 10 EID 50 / ml attenuated IBV; more preferably, the amount of attenuated IBV is 3 to 8 log 10 EID 50 / ml attenuated IBV; more preferably, the amount of attenuated IBV is 5 to 8 log 10 EID 50 / ml attenuated IBV; more preferably, the amount of attenuated IBV is 6 to 7 log 10 EID 50 / ml attenuated IBV; and most preferably, the amount of attenuated IBV is 6.5 log / ml. 10 EID 50 / ml attenuated IBV.

[0142] C2. The immunogenic composition or live attenuated vaccine of technical solution C1, which contains at least one veterinary acceptable vector; Preferably, the veterinary-acceptable carrier is a lyophilized protectant.

[0143] C3. An immunogenic composition or live attenuated vaccine of any one of technical solutions C1 to C2, wherein the immunogenic composition or live attenuated vaccine is formulated for single-dose administration; and / or The immunogenic composition or live attenuated vaccine is in the form of eye drops, nasal drops, ocular and nasal drops or spray, preferably in the form of a spray.

[0144] C4. An immunogenic composition or live attenuated vaccine of any one of technical solutions C1 to C3, wherein the immunogenic composition or live attenuated vaccine is used for the prevention and / or treatment of IBV infection in poultry, preferably poultry, more preferably chickens.

[0145] C5. An immunogenic composition or live attenuated vaccine of any one of technical solutions C1 to C4, wherein the poultry or chicken 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; preferably, the poultry or chicken is 1 day old.

[0146] Technical Solution Set D D1. A method for preparing an immunogenic composition or live attenuated vaccine against infectious bronchitis virus (IBV) infection, comprising the following steps: (1) Prepare attenuated IBV for any of the technical solutions in set A; (2) Add a veterinary-acceptable carrier, such as a lyophilized protectant, and mix to obtain an immunogenic composition or a live attenuated vaccine against IBV infection.

[0147] Technical Solution Collection E E1. A method for immunizing poultry, or for preventing or mitigating clinical signs of IBV in poultry, or for reducing ciliary arrest in poultry, wherein the method comprises administering to a subject a therapeutically or preventively effective amount of attenuated IBV from any of the techniques in set A, or a therapeutically or preventively effective amount of attenuated IBV prepared by any of the techniques in set B, or an immunogenic composition or attenuated live vaccine from any of the techniques in set C.

[0148] E2. Use of attenuated IBV of any technical method in set A, or attenuated IBV prepared by any technical method in set B, in the preparation of an immunogenic composition or attenuated live vaccine for immunizing poultry; preventing or preventing clinical signs caused by IBV in poultry; or for alleviating ciliary arrest in poultry.

[0149] E3. A therapeutically or preventively effective amount of attenuated IBV from any of the techniques in set A, or a therapeutically or preventively effective amount of attenuated IBV prepared by any of the techniques in set B, or a therapeutically or preventively effective amount of an immunogenic composition or attenuated live vaccine from any of the techniques in set C, or a therapeutically or preventively effective amount of an immunogenic composition or attenuated live vaccine prepared by any of the techniques in set D, used in methods for immunizing poultry, or for preventing or eliminating clinical signs caused by IBV in poultry, or for reducing ciliary arrest in poultry, wherein the method comprises administering the therapeutically or preventively effective amount of the attenuated IBV, immunogenic composition, or attenuated live vaccine to a subject.

[0150] E4. An immunogenic composition or attenuated live vaccine against infectious bronchitis virus (IBV) infection, comprising a therapeutically or preventively effective amount of attenuated IBV from any of the techniques in set A, or a therapeutically or preventively effective amount of attenuated IBV prepared by any of the techniques in set B, for use in methods of immunizing poultry, or in methods of preventing or avoiding clinical signs of IBV in poultry, or in methods of reducing ciliary arrest in poultry.

[0151] E5. Any of the methods or attenuated IBV, immunogenic compositions or attenuated live vaccines used in technical clauses E1 to E4, wherein the clinical signs caused by IBV are selected from the following: ciliary arrest, rales, decreased egg production, kidney damage, watery diarrhea, weight loss, viral load, viral shedding and combinations thereof; Preferably, the clinical sign caused by IBV is ciliary arrest; and / or Preferably, the poultry is domestic poultry, more preferably chicken; even more preferably, the chicken is 1 day old.

[0152] Example The following embodiments further illustrate the invention by way of example. It should be understood that the invention is not limited to any of the embodiments described below. Those skilled in the art will understand that, in view of the general description of the invention, the performance, results, and findings of these embodiments can be adapted and applied in a broader sense.

[0153] Example 1 - Passaging and attenuation of IB-QX K19 P7 strain In 2015, a research team from Shandong Agricultural University isolated a wild-type virus strain (named K19 or IB-QX K19 strain) from the kidney tissue of sick chickens in a suspected outbreak of infectious bronchitis at a chicken farm in Henan Province. Boehringer Ingelheim Animal Health (China) Co., Ltd. purchased the progeny of this wild-type strain after four generations of passage (4th generation), and then passaged this progeny continuously in chicken embryos up to the 7th generation. The 7th generation strain was identified as an avian IBV strain with the QX serotype, and this strain maintained high virulence. The strain passaged for 7 generations starting from the K19 strain was named IB-QX K19 P7 strain (IB-QX represents IBV with the QX serotype, and K19 P7 represents the progeny of the K19 strain after 7 generations of passage). Throughout this invention, the naming of similar virus strains has similar meanings.

[0154] The IB-QX K19 P7 strain was deposited on August 4, 2003, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan 430072, China, under accession number CCTCC NO: V202364.

[0155] The IB-QX K19 P7 strain was diluted 1:1000 with PBS and then inoculated into 8- to 10-day-old chicken embryos. Forty hours after infection, the embryos were refrigerated at 2-8°C for 24 hours, and then allantoic fluid was collected. Residual embryo tissue was removed by centrifugation to obtain the 8th generation virus strain (named IB-QX K19 P8 strain). Virulence testing showed that the IB-QX K19 P8 strain remained highly virulent. The IB-QX K19 P8 strain was used as a challenge strain in the following examples to evaluate the protective efficacy against a range of attenuated strains. The IB-QX K19 P8 strain was deposited on August 4, 2003, at CCTCC, Wuhan University, Wuhan 430072, China, under accession number CCTCC NO: V202365.

[0156] The IB-QX K19 P8 strain was continuously passaged using the method described above, and virus strains of the 162nd, 171st, and 179th generations were prepared, respectively. The safety test results in Example 2 showed that the virulence of the 162nd generation strain was significantly reduced without causing any IB-related clinical symptoms in chickens. All three virus strains are attenuated IBV strains, named IB-QX K19-A P162, IB-QX K19-A P171, and IB-QX K19-A P179 strains, respectively ("A" stands for "attenuated"). Throughout this invention, the naming of similar attenuated strains has a similar meaning.

[0157] The IB-QX K19-A P162 strain was deposited on August 4, 2003, at CCTCC, Wuhan University, Wuhan 430072, China, under accession number CCTCC NO: V202366. The nucleotide sequence of the S1 gene of the IB-QX K19-A P162 strain is described in SEQ ID NO:1; and the amino acid sequence encoded by the S1 gene of the IB-QX K19-A P162 strain (S1 protein) is described in SEQ ID NO:4.

[0158] SEQ ID NO:1 SEQ ID NO:4 MLGKSLFLVTILCALCSANLFDSANNYVYYYQSAFRPPNGWHLQGGAYAVVNSTNYTNNAGSASQCTVGVIKDVYNQSAASIAMTAPLQGMAWSKSQFCSAHCNFSEITVFVTHCYSSGAGSCPITGMIPRDHI RISAMKNGSLFYNLTVSVSKYPNFKSFQCVNNFTSVYLNGDLVFTSXKTTDVTSAGVYFKAGGPVNYSIMKEFKVLAYFVNGTAQDVILCDKSPKGLLACQYNTGNFSDGFYPFTNTTLVREKFTVYRESSVNTT LALTNFTFTNVSNAQPNSGGVNTFHLYQTQTAQSGYYNFNLSFLSQFVYKASDFMYGSYHPSCSFRPETINSGLWFNSLSVSLAYGPLQGGCKQSVFSGRATCCYAYSYNGPIACKGVYAGELRTNFECGLLIYV TKSDGSRIQIRTEEPLVLTQHNYNNITLDKCVEYNIYGRVGQGFITNVTDSAANFSYLADGGLAILDTSGAIDVFVVQGSYGLNYYKVNPCEDVNQQFVVSGGNIVGILTSRNETGSEQVENQFYVKLTNSSHRRR The IB-QX K19-A P171 strain was deposited on August 4, 2003, at CCTCC, Wuhan University, Wuhan 430072, China, under accession number CCTCC NO: V202367. The nucleotide sequence of the S1 gene of the IB-QX K19-A P171 strain is described in SEQ ID NO:2; and the amino acid sequence encoded by the S1 gene of the IB-QX K19-A P171 strain (S1 protein) is described in SEQ ID NO:5.

[0159] SEQ ID NO:2 SEQ ID NO:5 MLGKSLFLVTILCALCSANLFDSANNYVYYYQSAFRPPNGWHLQGGAYAVVNSTNYTNNAGSASQCTVGVIKDVYNQSAASIAMTAPLQGMAWSKSQFCSAHCNFSEITVFVTHCYSSGAGSCPITGMIPRDHI RISAMKNGSLFYNLTVSVSKYPNFKSFQCVNNFTSVYLNGDLVFTSTKTTDVTSAGVYFKAGGPVNYSIMKEFKVLAYFVNGTAQDVILCDKSPKGLLACQYNTGNFSDGFYPFTNTTLVREKFTVYRESSVNTT LALTNFTFTNVSNAQPNSGGVNTFHLYQTQTAQSGYYNFNLSFLSQFVYKASDFMYGSYHPSCSFRPETINSGLWFNSLSVSLAYGPLQGGCKQSVFSGRATCCYAYSYNGPIACKGVYAGELRTNFECGLLIYV TKSDGSRIQIRTEEPLVLTQHNYNNITLDKCVEYNIYGRVGQGFITNVTDSAANFSYLADGGLAILDTSGAIDVFVVQGSYGLNYYKVNPCEDVNQQFVVSGGNIVGILTSRNETGSEQVENQFYVKLTNSSHRRR The IB-QX K19-A P179 strain was deposited on August 4, 2003, at CCTCC, Wuhan University, Wuhan 430072, China, under accession number CCTCC NO: V202368. The nucleotide sequence of the S1 gene of the IB-QX K19-A P179 attenuated strain is described in SEQ ID NO:3; and the amino acid sequence encoded by the S1 gene of the IB-QX K19-A P179 strain (S1 protein) is described in SEQ ID NO:6.

[0160] SEQ ID NO:3 SEQ ID NO:6 MLGKSLFLVTILCALCSANLFDSANNYVYYYQSAFRPPNGWHLQGGAYAVVNSTNYTNNAGSASQCTVGVIKDVYNQSAASIAMTAPLQGMAWSKSQFCSAHCNFSEITVFVTHCYSSGAGSCPITGMIPRDHI RISAMKNGSLFYNLTVSVSKYPNFKSFQCVNNFTSVYLNGDLVFTSTKTTDVTSAGVYFKAGGPVNYSIMKEFKVLAYFVNGTAQDVILCDKSPKGLLACQYNTGNFSDGFYPFTNTTLVREKFTVYRESSVNTT LALTNFTFTNVSNAQPNSGGVNTFHLYQTQTAQSGYYNFNLSFLSQFVYKASDFMYGSYHPSCSFRPETINSGLWFNSLSVSLAYGPLQGGCKQSVFSGRATCCYAYSYNGPIACKGVYAGELRTNFECGLLIYV TKSDGSRIQIRTEEPLVLTQHNYNNITLDKCVEYNIYGRVGQGFITNVTDSAANFSYLADGGLAILDTSGAIDVFVVQGSYGLNYYKVNPCEDVNQQFVVSGGNIVGILTSRNETGSEQVENQFYVKLTNSSHRRR The S1 gene sequences of two strains each of IB-QX K19-A P162, IB-QX K19-A P171, and IB-QX K19-A P179 were sequentially aligned. Sequence alignment was performed using DNAMAN software with default parameters. The results showed that the sequence identity of the S1 gene between IB-QX K19-A P162 and IB-QX K19-A P171 was 99.9%, the sequence identity between IB-QX K19-A P162 and IB-QX K19-A P179 was 99.8%, and the sequence identity between IB-QX K19-A P171 and IB-QX K19-A P179 was 99.9% (see [link to original text]). Figure 1-3The sequence identity of the S1 protein between strains IB-QX K19-A P162 and IB-QX K19-A P171 is 99.8%, the sequence identity of the S1 protein between strains IB-QX K19-A P162 and IB-QX K19-A P179 is 99.8%, and the sequence identity of the S1 protein between strains IB-QX K19-A P171 and IB-QX K19-A P179 is 100.0% (see [link to original text]). Figure 4-6 Based on the results, it can be determined that the nucleotide sequence of the S1 gene / protein remained almost unchanged over a range of approximately 20 generations, from generation 162 to generation 179.

[0161] Example 2 - Safety and virulence reversion assessment of IB-QX K19-A P162 strain One-day-old SPF chickens were vaccinated via ocular and nasal routes using the IB-QX K19-A P162 strain. Tracheal tissue from SPF chickens was collected five days post-vaccination and prepared as tissue homogenates. One-day-old SPF chickens were vaccinated using these tissue homogenates, and the safety of the IB-QX K19-A P162 strain in one-day-old SPF chickens and its virulence reversion after five passages were evaluated.

[0162] The IB-QX K19-A P162 strain was passaged 5 times in SPF chickens. Except for the fifth passage which used 10 SPF chickens, 16 SPF chickens were used in each of the first to fourth passages.

[0163] First passaging: 1-day-old SPF chickens were administered the virus via ocular and nasal route using 50 μl of the IB-QX K19-A P162 strain at 10... 5.5 EID 50 Chickens were inoculated with a single dose, thus completing the first passage (the strain following the first passage, starting with the IB-QX K19-AP162 strain, was named P162-BP1). Ten SPF chickens in this group were clinically observed for a total of 10 days (Group 1).

[0164] Five days after vaccination, tracheal tissue was collected from the remaining six SPF chickens and prepared into tissue homogenates as inoculum for the next passage. The inoculum was then subjected to virus isolation and identification to ensure successful passage.

[0165] The preparation method for the tissue homogenate is as follows: Approximately 1.5 cm of fresh tracheal tissue was obtained from each SPF chicken and placed in a sterile centrifuge tube. The tissue sample was cut into small pieces and ground with beads, and 1 ml of PBS containing 10% antibiotics was added. The sample was homogenized using a tissue homogenizer at a frequency of 30 times / second for 5 minutes; then the sample was centrifuged at 12000 rpm for 5 minutes, and the supernatant was collected. Tracheal tissue homogenates from 6 SPF chickens in each group were combined and used as inoculum for the next passage.

[0166] Second, third, and fourth passages: One-day-old SPF chickens were inoculated via the ocular and nasal routes with 50 μl of tracheal tissue homogenate prepared from previous generations (single dose), thus completing the second, third, and fourth passages (the strains after the second, third, and fourth passages were named P162-BP2, P162-BP3, and P162-BP4, respectively). Ten SPF chickens from each generation were selected for clinical observation for a total of 10 days (groups 2, 3, and 4).

[0167] Similarly, tracheal tissue was collected from the remaining 6 SPF chickens 5 days after vaccination and then prepared into tissue homogenates as inoculum for the next passage (the preparation method of the tissue homogenates was the same as that of the first passage).

[0168] Fifth passage: One-day-old SPF chickens were inoculated via the ocular and nasal routes with 50 μl of tracheal tissue homogenate prepared from the fourth passage. Ten SPF chickens in this group were then clinically observed for a total of 21 days (Group 5).

[0169] A control group and a negative control group (i.e., groups 6 and 7) of the IB-QX K19-A P162 strain were also set up, with 10 SPF chickens in each group. The SPF chickens in group 6 were administered the IB-QX K19-A P162 strain via the ocular and nasal routes at 10... 5.5 EID 50 The chickens were vaccinated at the prescribed dose. SPF chickens in group 7 were administered phosphate-buffered saline (PBS) via the ocular and nasal routes. Each group of SPF chickens underwent clinical observation for a total of 21 days.

[0170] Table 1: Experimental Design of Example 2

[0171] "-" means "unavailable" The results showed that after 1-day-old chickens were inoculated with the IB-QX K19-A P162 strain, and the tracheal tissue prepared from each subsequent generation contained more than 10 4.63 EID 50The viral load is 1 / mL. The IB-QX K19-A P162 strain exhibits excellent adaptability to chickens and can be stably passaged in 1-day-old chickens.

[0172] Table 2: Viral load in each inoculum

[0173] The results also showed that 1-day-old chickens inoculated with the IB-QX K19-A P162 strain did not exhibit any IB-related symptoms or mortality. SPF chickens vaccinated with subsequent inoculums did not show any IB-related symptoms, and no pathological changes were found in target organs during necropsy. The morbidity and mortality rates in SPF chickens across each of the five passages were both 0%. The IB-QX K19-A P162 strain did not show any reversion to virulence after five passages. Based on these results, it can be determined that the IB-QXK19-A P162 strain and its offspring within five passages are safe for 1-day-old chickens.

[0174] Example 3 - Efficacy evaluation of IB-QX K19-A P162 and IB-QX K19P171 strains via ocular and nasal routes The protective efficacy of IB-QX K19-A P162 and IB-QX K19-A P171 strains against IBV infection was evaluated.

[0175] One hundred one-day-old SPF chickens were randomly divided into four groups of 25 each. Groups 1 and 2 served as the immunization groups, while group 3 served as the challenge control group. Twenty-five one-day-old SPF chickens from the same source and in separate batches served as the control group (group 4), without vaccination or infection.

[0176] In groups 1 and 2, SPF chickens were administered 50 μl of IB-QX K19-A P162 strain and IB-QXK19-A P171 strain (10 μl each) via ocular and nasal routes, respectively. 4.0 EID 50 Chickens were vaccinated (single dose). SPF chickens in group 3 were administered 50 μl of PBS via ocular and nasal route as a challenge control group.

[0177] Table 3: Experimental Design of Example 3

[0178] "-" means "unavailable" From the day of vaccination until the day before the attack, all SPF chickens were subjected to daily clinical observation to record whether the SPF chickens had any IB-related symptoms such as coughing, sneezing, difficulty breathing, head shaking, or tear stains.

[0179] The attack was performed 21 days post-vaccination. SPF chickens in groups 1-3 were administered 50 μl of the IB-QXK19 P8 strain via the ocular and nasal route at 10... 4.0 EID 50 Chickens were challenged with a dose specified for SPF. SPF chickens were observed for 5 consecutive days after the challenge.

[0180] Following chicken challenge, all SPF chickens underwent daily clinical observation and scoring to record any IB-related symptoms. Scoring criteria: Normal = 0 points, and Abnormal = 1 point.

[0181] Five days after the attack, all surviving SPF chickens were euthanized, and fresh tracheal tissue samples were collected from each chicken. Tracheal tissue from five SPF chickens in each group was selected to prepare tracheal rings for ciliary arrest scoring.

[0182] Rating system: 0 = 100% tracheal rings show ciliary activity.

[0183] 1 = More than 75% but less than 100% of the tracheal rings show ciliary activity.

[0184] 2 = More than 50% but less than 75% of the tracheal rings show ciliary activity.

[0185] 3 = More than 25% but less than 50% of the tracheal rings show ciliary activity.

[0186] 4 = Less than 25% of the tracheal rings show ciliary activity or do not show ciliary activity.

[0187] If the score of two or more tracheal rings is >2, the chicken is identified as infected with IBV (IBV positive).

[0188] During the experiment, clinical observations were conducted daily, and groups 1-5 were observed and recorded for 27 consecutive days.

[0189] Following vaccination, none of the SPF chickens in each group showed clinical abnormalities and were in good health.

[0190] No clinical abnormalities were observed in SPF chickens in groups 1-2 after challenge, and no gross pathological changes were observed at necropsy. Tracheal tissue samples were collected 5 days post-challenge, and IBV was isolated from them. The positivity rates of tracheal samples from SPF chickens in groups 1-3 were 10% (2 / 20), 5% (1 / 20), and 100% (20 / 20), respectively. The protection rates of the IB-QX K19-A P162 strain and the IB-QX K19-A P171 strain against IBV infection in groups 1-2 were 90% (2 / 20) and 95% (1 / 20), respectively.

[0191] Table 4: Protection Rate in Each Group

[0192] Note 1: "-" means "unavailable".

[0193] Note 2: “a” shows the clinical observation results after the attack; “b” shows the pathological observation results visible to the naked eye during the autopsy; and “c” shows the positivity rate of the virus.

[0194] Five days after the attack, the tracheal ring ciliary score was performed on SPF chickens in groups 1-3. The results showed that the number of SPF chickens with a tracheal ring score >2 was 0 / 5, 0 / 5, and 5 / 5, respectively. The morbidity rate of SPF chickens in each group was 0%, 0%, and 100%, respectively.

[0195] Based on the results above, it can be determined that the IB-QX K19-A P162 and IB-QX K19-A P171 strains can provide excellent protection against IBV infection in SPF chickens.

[0196] Example 4 - Efficacy evaluation of IB-QX K19-A P162, IB-QX K19-A P171, and IB-QX K19P179 strains via spray route The protective efficacy of IB-QX K19-A P162, IB-QX K19-A P171, and IB-QX K19P179 strains against IBV infection was evaluated.

[0197] One hundred and twenty one-day-old SPF chickens were randomly divided into four groups of 30 each. Groups 1-3 served as the immunization group, and group 4 served as the challenge control group.

[0198] SPF chickens in groups 1-3 were treated by spraying with IB-QX K19-A P162, IB-QX K19-A P171, and IB-QX K19P179 strains, respectively (10...). 5.0 EID 50Chickens were vaccinated (single dose). Group 4 SPF chickens were administered PBS via a spray route as a challenge control group.

[0199] Table 5: Experimental Design of Example 4

[0200] Note 1: "-" means "unavailable".

[0201] From the day of vaccination until the day before the attack, all SPF chickens were subjected to daily clinical observation to record whether the SPF chickens had any IB-related symptoms such as coughing, sneezing, difficulty breathing, head shaking, or tear stains.

[0202] The attack was performed 21 days post-vaccination. SPF chickens in groups 1-4 were administered the IB-QX K19 P8 strain via nasal drop at a dose of 10... 4.0 EID 50 / Chicken dose (50 μl) was used for attack.

[0203] Following chicken challenge, all SPF chickens underwent daily clinical observation and scoring to record any IB-related symptoms. Scoring criteria: Normal = 0 points, and Abnormal = 1 point.

[0204] Five days post-challenge, 20 SPF chickens from each group were selected and euthanized. Fresh tracheal tissue samples were collected from each chicken for virus isolation. Six days post-challenge, the remaining 10 SPF chickens from each group were euthanized, and their tracheal tissue was collected to prepare tracheal rings for ciliary arrest scoring. The criteria for ciliary arrest scoring were the same as in Example 3.

[0205] The results showed that the positive rate of virus isolation from tracheal tissue samples in group 4 (control group) was 90% (18 / 20), while the positive rate in groups 1-3 was 10% (2 / 20). The protection rates of IB-QX K19-A P162, IB-QX K19-A P171, and IB-QX K19-A P179 strains against IBV infection in SPF chickens in groups 1-3 were all 90% (18 / 20).

[0206] Table 6: Protection rates of the three attenuated strains

[0207] The results also showed that the number of SPF chickens with a tracheal ring score >2 in groups 1-4 were 0 / 10, 2 / 10, 0 / 10, and 10 / 10, respectively. The morbidity rates of SPF chickens in each group were 0%, 20%, 0%, and 100%, respectively.

[0208] Table 7: Morbidity rate in SPF chickens

[0209] The results confirmed that strains IB-QX K19-A P162, IB-QX K19-A P171, and IB-QX K19-A P179 all provide sufficient protection against IBV in 1-day-old chickens, and all three attenuated strains can be used to develop vaccines against IBV infection. Furthermore, the efficacy of the three attenuated strains meets the requirements for large-scale spray immunization in chicken farms. Compared to the ocular and nasal routes, spray immunization requires significantly less manpower and greatly shortens the vaccination time.

[0210] The results also confirmed that the IB-QX K19-AP171 and IB-QX K19P179 strains maintained excellent immunoprotective efficacy against IBV infection for at least approximately 20 generations starting from the IB-QX K19-A P162 strain. That is, a wide range of progeny can be derived from the IB-QX K19-A P162 and IB-QX K19-A P171 strains, and these progeny all effectively provide immunoprotection against IBV infection. Unbound by any theoretical constraints, the wide range of progeny likely stems from the high sequence identity in the S1 gene / protein sequence from the IB-QX K19-A P162 strain to the IB-QX K19-A P179 strain. This wide range of progeny ensures a broader passage window during production, thus facilitating large-scale industrial production.

[0211] Example 5 – Efficacy evaluation of the IB-QX K19-A P162 strain at lower vaccine doses The protective efficacy of the IB-QX K19-A P162 strain against IBV infection was evaluated at lower vaccine doses.

[0212] Sixty one-day-old SPF chickens were randomly divided into five groups of 12 each. Groups 1-3 served as the immunization group, group 4 as the challenge control group, and group 5 as the negative control group. The SPF chickens in groups 1-3 were sprayed with the IB-QXK19-A P162 strain at a concentration of 10 mg / L. 5.0 EID 50 / Chicken, 10 4.0 EID 50 / Chicken and 10 3.0 EID 50 Vaccination was administered to chickens at a single dose. SPF chickens in groups 4-5 were vaccinated with PBS via a spray route.

[0213] The attack was performed 21 days post-vaccination. SPF chickens in groups 1-4 were administered the IB-QX K19 P8 strain via nasal drop at a dose of 10... 4.0 EID 50 Chickens were challenged with a dose of 50 μl. Five days post-challenge, all SPF chickens from each group were euthanized. Fresh tracheal tissue samples were collected from each chicken to prepare tracheal rings for ciliary arrest scoring. The criteria for ciliary arrest scoring were the same as in Example 3.

[0214] Table 8: Experimental Design of Example 5

[0215] "-" means "unavailable" The results showed that when its dose was reduced to 10 3.0 EID 50 When used in chickens, the IB-QX K19-A P162 strain still provides sufficient protection against IBV infection in 1-day-old chickens via spraying. This further confirms that the IB-QX K19-A P162 strain has excellent immunogenicity and can provide comprehensive protection against IBV infection in chickens.

[0216] Sequence List: Nucleotide sequence of the S1 gene of SEQ ID NO:1 - IB-QX K19-A P162 strain Nucleotide sequence of the S1 gene of SEQ ID NO:2 - IB-QX K19-A P171 strain Nucleotide sequence of the S1 gene of SEQ ID NO:3- IB-QX K19-A P179 strain Amino acid sequence of S1 protein of SEQ ID NO:4 - IB-QX K19-A P162 strain Amino acid sequence of S1 protein of SEQ ID NO:5 - IB-QX K19-A P171 strain Amino acid sequence of S1 protein of SEQ ID NO:6 - IB-QX K19-A P179 strain PCT / RO / 134 form

Claims

1. An attenuated infectious bronchitis virus (IBV), wherein the attenuated IBV comprises an S1 gene containing or composed of a nucleotide sequence, the nucleotide sequence encoding an amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO:4; Preferably, the attenuated IBV comprises an S1 gene containing or composed of nucleotide sequences, the nucleotide sequences encoding an amino acid sequence having at least 96%, 97%, 98%, 99% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO:4; More preferably, the attenuated IBV comprises an S1 gene containing or composed of nucleotide sequences, the nucleotide sequences encoding an amino acid sequence having at least 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO:4; or The attenuated IBV includes an S1 gene comprising or composed of nucleotide sequences, the nucleotide sequences encoding amino acid sequences selected from the following: SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:

6.

2. The attenuated IBV of claim 1, wherein the S1 gene comprises or is composed of a nucleotide sequence, the nucleotide sequence having at least 95% sequence identity with the nucleotide sequence shown in SEQ ID NO:1; Preferably, the S1 gene comprises or is composed of nucleotide sequences, wherein the nucleotide sequences have at least 96%, 97%, 98%, 99% or 100% sequence identity with the nucleotide sequence shown in SEQ ID NO:1; More preferably, the S1 gene comprises or is composed of a nucleotide sequence, the nucleotide sequence having at least 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% sequence identity with the nucleotide sequence shown in SEQ ID NO:1; or The S1 gene contains or consists of nucleotide sequences selected from the following: SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:

3.

3. The attenuated IBV of claim 1 or 2, wherein the attenuated IBV has the QX serotype; preferably, wherein the attenuated IBV is derived from the IB-QX K19 P7 strain deposited under accession number CCTCCNO: V202364.

4. The attenuated IBV of any one of claims 1 to 3, wherein the attenuated IBV is at least the 160th generation; preferably, the 160th to 200th generation; more preferably, the 160th to 180th generation; even more preferably, the 162nd to 179th generation; even more preferably, the 162nd, 163rd, 164th, 165th, 166th, 167th, 168th, 169th, 170th, 171st, 172nd, 173rd, 174th, 175th, 176th, 177th, 178th or 179th generation; and most preferably, the 162nd, 171st or 179th generation.

5. An attenuated infectious bronchitis virus (IBV), wherein the attenuated IBV can be obtained by passage of the IB-QX K19 P7 strain deposited under accession number CCTCC NO: V202364 to at least 160 generations; preferably 160 to 200 generations; more preferably 160 to 180 generations; more preferably 162 to 179 generations; more preferably 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178 or 179 generations; and most preferably 162, 171 or 179 generations.

6. The attenuated IBV of claim 5, wherein the attenuated IBV comprises an S1 gene containing or composed of nucleotide sequences, the nucleotide sequences encoding an amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO:4; Preferably, the attenuated IBV comprises an S1 gene containing or composed of nucleotide sequences, the nucleotide sequences encoding an amino acid sequence having at least 96%, 97%, 98%, 99% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO:4; More preferably, the attenuated IBV comprises an S1 gene containing or composed of nucleotide sequences, the nucleotide sequences encoding an amino acid sequence having at least 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO:4; or The attenuated IBV includes an S1 gene comprising or composed of nucleotide sequences, the nucleotide sequences encoding amino acid sequences selected from the following: SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:

6.

7. The attenuated IBV of claim 5 or 6, wherein the S1 gene comprises or is composed of a nucleotide sequence, the nucleotide sequence having at least 95% sequence identity with the nucleotide sequence shown in SEQ ID NO:1; Preferably, the S1 gene comprises or is composed of nucleotide sequences, wherein the nucleotide sequences have at least 96%, 97%, 98%, 99% or 100% sequence identity with the nucleotide sequence shown in SEQ ID NO:1; More preferably, the S1 gene comprises or is composed of a nucleotide sequence, the nucleotide sequence having at least 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% sequence identity with the nucleotide sequence shown in SEQ ID NO:1; or The S1 gene contains or consists of nucleotide sequences selected from the following: SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:

3.

8. An attenuated infectious bronchitis virus (IBV). The attenuated IBV mentioned therein refers to the IB-QX K19-A P162 strain and its progeny deposited under accession number CCTCC NO: V202366, and the progeny of the IB-QX K19-A P162 strain is any attenuated progeny IBV possessing all the identifying characteristics of the deposited IB-QX K19-A P162 strain. These identifying characteristics include, for example, an S1 protein identical to that of the IB-QX K19-A P162 strain shown in SEQ ID NO:4, or an S1 protein identical to that of the IB-QX K19-A P162 strain shown in SEQ ID NO:

4. The S1 protein of strain P162 has at least 99%, preferably at least 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% sequence identity, and / or protection against virulent IB-QX attack, including protection against virulent IB-QX attack in at least 80%, preferably 85%, more preferably 90%, more preferably 95% and most preferably 100% of poultry, or improved ciliary stagnation score after virulent IB-QX attack in at least 90%, preferably 95% and most preferably 100% of poultry; The attenuated IBV mentioned therein refers to the IB-QX K19-A P171 strain and its progeny deposited under accession number CCTCC NO: V202367, and the progeny of the IB-QX K19-A P171 strain is any attenuated progeny IBV possessing all the identifying characteristics of the deposited IB-QX K19-A P171 strain. These identifying characteristics include, for example, an S1 protein identical to that of the IB-QX K19-A P171 strain shown in SEQ ID NO:5, or an S1 protein identical to that of the IB-QX K19-A P171 strain shown in SEQ ID NO:

5. The S1 protein of strain P171 has at least 99%, preferably at least 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% sequence identity, and / or protection against virulent IB-QX attack, including protection against virulent IB-QX attack in at least 80%, preferably 85%, more preferably 90%, more preferably 95%, and most preferably 100% of poultry, or improved ciliary stagnation score after virulent IB-QX attack in at least 90%, preferably 95%, and most preferably 100% of poultry; or The attenuated IBV mentioned therein refers to the IB-QX K19-A P179 strain and its progeny deposited under accession number CCTCC NO: V202368, and the progeny of the IB-QX K19-A P179 strain is any attenuated progeny IBV possessing all the identifying characteristics of the deposited IB-QX K19-A P179 strain. These identifying characteristics include, for example, an S1 protein identical to that of the IB-QX K19-A P179 strain shown in SEQ ID NO: 6, or an S1 protein identical to that of the IB-QX K19-A P179 strain shown in SEQ ID NO:

6. The S1 protein of strain P179 has at least 99%, preferably at least 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% sequence identity, and / or protection against virulent IB-QX attack, including protection against virulent IB-QX attack in at least 80%, preferably 85%, more preferably 90%, more preferably 95% and most preferably 100% of poultry, or improved ciliary stagnation score after virulent IB-QX attack in at least 90%, preferably 95% and most preferably 100% of poultry.

9. The attenuated IBV of claim 8, wherein - The offspring of the IB-QX K19-A P162 strain have the same S1 gene as the IB-QX K19-A P162 strain shown in SEQ ID NO:1, or have an S1 gene with at least 99%, preferably at least 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% sequence identity as the S1 gene of the IB-QX K19-A P162 strain shown in SEQ ID NO:1; - The progeny of the IB-QX K19-A P171 strain possess the same S1 gene as the IB-QX K19-A P162 strain shown in SEQ ID NO:2, or have an S1 gene with at least 99%, preferably at least 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% sequence identity as the S1 gene of the IB-QX K19-A P162 strain shown in SEQ ID NO:2; or - The offspring of the IB-QX K19-A P179 strain have the same S1 gene as the IB-QX K19-A P162 strain shown in SEQ ID NO:3, or have an S1 gene with at least 99%, preferably at least 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% sequence identity as the S1 gene of the IB-QX K19-A P162 strain shown in SEQ ID NO:

3.

10. The attenuated IBV of claim 8 or 9, wherein - The offspring of the IB-QX K19-A P162 strain include at most 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 generations of the IB-QX K19-A P162 strain; or the offspring of the IB-QX K19-A P162 strain are the 10th, 9th, 8th, 7th, 6th, 5th, 4th, 3rd, 2nd, or 1st generation of the IB-QX K19-A P162 strain; or the offspring of the IB-QX K19-AP162 strain can be obtained by passing the IB-QX K19-A P162 strain through 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 generation; preferably, the offspring of the IB-QX K19-A P162 strain can be obtained by passing the IB-QX K19-A P162 strain through 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 generation of the IB-QX K19-A P162 strain. P162 strain is obtained by passing it through 5, 4, 3, 2 or 1 generations; - The offspring of the IB-QX K19-A P171 strain include at most 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 generations of the IB-QX K19-A P171 strain; or the offspring of the IB-QX K19-A P171 strain are the 10th, 9th, 8th, 7th, 6th, 5th, 4th, 3rd, 2nd, or 1st generation of the IB-QX K19-A P171 strain; or the offspring of the IB-QX K19-AP171 strain can be obtained by passing the IB-QX K19-A P171 strain through 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 generation; preferably, the offspring of the IB-QX K19-A P171 strain can be obtained by passing the IB-QX K19-A P171 strain through 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 generation of the IB-QX K19-A P171 strain. P171 strain was obtained by passage 5, 4, 3, 2 or 1 times; or - The offspring of the IB-QX K19-A P179 strain include at most 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 generations of the IB-QX K19-A P179 strain; or the offspring of the IB-QX K19-A P179 strain are the 10th, 9th, 8th, 7th, 6th, 5th, 4th, 3rd, 2nd, or 1st generation of the IB-QX K19-A P179 strain; or the offspring of the IB-QX K19-AP179 strain can be obtained by passing the IB-QX K19-A P179 strain through 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 generation; preferably, the offspring of the IB-QX K19-A P179 strain can be obtained by passing the IB-QX K19-A P179 strain through 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 generation of the IB-QX K19-A P179 strain. P179 strain is obtained by passing it through 5, 4, 3, 2 or 1 generations.

11. An attenuated infectious bronchitis virus (IBV), wherein the attenuated IBV is selected from: the IB-QX K19-A P162 strain deposited under accession number CCTCC NO: V202366, any of the progeny of the IB-QX K19-A P162 strain comprising up to 5, 4, 3, 2, or 1 generations, the IB-QX K19-A P171 strain deposited under accession number CCTCC NO: V202367, any of the progeny of the IB-QX K19-A P171 strain comprising up to 5, 4, 3, 2, or 1 generations, the IB-QX K19-A P179 strain deposited under accession number CCTCC NO: V202368, and the IB-QX K19-A P179 strain comprising IB-QX K19-A P171. Any of the offspring of the IB-QXK19-A P179 strain at most 5, 4, 3, 2 or 1 generations.

12. The attenuated IBV of any one of claims 1-11, wherein the attenuated IBV is isolated.

13. A method for preparing attenuated infectious bronchitis virus (IBV), comprising the following steps: The IBV strain is passaged in chicken embryos or chicken embryo-derived cells for no more than 10 generations; more preferably, the IBV strain is passaged in chicken embryos or chicken embryo-derived cells for 5, 4, 3, 2 or 1 generations, wherein the IBV strain is selected from the IB-QX K19-A P162 strain deposited under accession number CCTCC NO: V202366, the IB-QX K19-AP171 strain deposited under accession number CCTCC NO: V202367, and the IB-QX K19-A P179 strain deposited under accession number CCTCC NO: V202368.

14. The method of claim 13, wherein the chicken embryo-derived cells are primary chicken embryo cells; more preferably, the primary chicken embryo cells are fibroblasts or cells derived from kidney, liver or lung tissue.

15. An immunogenic composition or live attenuated vaccine against infectious bronchitis virus (IBV) infection, comprising a therapeutically or preventively effective amount of attenuated IBV of any one of claims 1 to 12, or a therapeutically or preventively effective amount of attenuated IBV prepared by the method of any one of claims 13 to 14. Preferably, the amount of attenuated IBV is 1 to 10 log. 10 EID 50 / ml attenuated IBV; more preferably, the amount of attenuated IBV is 2 to 8 log 10 EID 50 / ml attenuated IBV; more preferably, the amount of attenuated IBV is 3 to 8 log 10 EID 50 / ml attenuated IBV; more preferably, the amount of attenuated IBV is 5 to 8 log 10 EID 50 / ml attenuated IBV; more preferably, the amount of attenuated IBV is 6 to 7 log 10 EID 50 / ml attenuated IBV; and most preferably, the amount of attenuated IBV is 6.5 log / ml. 10 EID 50 / ml attenuated IBV.

16. The immunogenic composition or live attenuated vaccine of claim 15, comprising at least one veterinary acceptable carrier; Preferably, the veterinary-acceptable carrier is a lyophilized protectant.

17. The immunogenic composition or live attenuated vaccine of any one of claims 15 to 16, wherein the immunogenic composition or live attenuated vaccine is formulated for single-dose administration; and / or The immunogenic composition or live attenuated vaccine is in the form of eye drops, nasal drops, ocular and nasal drops or spray, preferably in the form of a spray.

18. The immunogenic composition or live attenuated vaccine of any one of claims 15 to 17, wherein the immunogenic composition or live attenuated vaccine is used for the prevention and / or treatment of IBV infection in poultry, preferably poultry, more preferably chickens.

19. The immunogenic composition or live attenuated vaccine of any one of claims 15 to 18, wherein the poultry or chicken 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; preferably, the poultry or chicken is 1 day old.

20. A method for immunizing poultry, or a method for preventing or avoiding clinical signs caused by IBV in poultry, or a method for reducing ciliary stagnation in poultry, wherein the method comprises administering to a subject a therapeutically or preventively effective amount of attenuated IBV of any one of claims 1 to 12, or a therapeutically or preventively effective amount of attenuated IBV prepared by the method of any one of claims 13 to 14, or a therapeutically or preventively effective amount of an immunogenic composition or attenuated live vaccine of any one of claims 15 to 19.

21. A therapeutically or preventively effective amount of attenuated IBV of any one of claims 1 to 12, or a therapeutically or preventively effective amount of attenuated IBV prepared by the method of any one of claims 13 to 14, or a therapeutically or preventively effective amount of an immunogenic composition or attenuated live vaccine of any one of claims 15 to 19, used in methods for immunizing poultry, or for preventing or avoiding clinical signs caused by IBV in poultry, or for reducing ciliary arrest in poultry, wherein the method comprises administering the therapeutically or preventively effective amount of the attenuated IBV, immunogenic composition, or attenuated live vaccine to a subject.

22. The method of claim 20 or the attenuated IBV, immunogenic composition or attenuated live vaccine used for the purpose of claim 21, wherein the clinical signs caused by IBV are selected from the following: ciliary arrest, rales, decreased egg production, kidney damage, watery diarrhea, weight loss, viral load, viral shedding and combinations thereof; Preferably, the clinical sign caused by IBV is ciliary arrest; and / or Preferably, the poultry is domestic poultry, more preferably chicken; even more preferably, the chicken is 1 day old.

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