WO2025055902A1

WO2025055902A1 - Attenuated infectious bronchitis virus and a vaccine comprising the same

Info

Publication number: WO2025055902A1
Application number: PCT/CN2024/118017
Authority: WO
Inventors: Chao TONG; Ning Chen; Huanhuan LIU; Rongchun WU
Original assignee: Boehringer Ingelheim Vetmedica China Co Ltd
Current assignee: Boehringer Ingelheim Vetmedica China Co Ltd
Priority date: 2023-09-11
Filing date: 2024-09-10
Publication date: 2025-03-20
Anticipated expiration: 2026-03-11

Abstract

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

Description

Attenuated infectious bronchitis virus and a vaccine comprising the same

Cross-Reference to Related Application

This application claims the priority of PCT/CN2023/117927 filed on September 11, 2023 entiled by “Attenuated infectious bronchitis virus and a vaccine comprising the same” , the entirety of which is incorporated by reference herein.

Field of the Invention

The present invention relates to the field of animal health. In particular, the invention relates to an attenuated infectious bronchitis virus and an attenuated live vaccine comprising the same. The invention also relates to a method of preparing an attenuated infectious bronchitis virus, and a method of immunizing birds by administrating the vaccine.

Background of the Invention

Infectious bronchitis (IB) is an acute and highly contagious infection caused by infectious bronchitis virus (IBV) , which is one of the most serious viral infections in poultry industry. There are many serotypes of IBVs. Since the mid-1990s, new serotype strains of IBVs have been emerging in different regions. IBVs of different serotypes can only provide partial cross immune protection or even no cross immune protection. Since 1996, IB caused by IBVs of serotype QX (IB-QX) has become the epidemic disease in China. It is needed to develop a vaccine against IB-QX.

Brief Description of the Invention

In one aspect, the invention provides an attenuated IBV, wherein the attenuated IBV comprises a S1 gene comprising or consisting of a nucleotide sequence encoding an amino acid sequence having at least 95%sequence identity to the amino acid sequence set forth in SEQ ID NO: 4; or the attenuated IBV comprises a S1 gene comprising or consisting of a nucleotide sequence encoding an amino acid sequence selected from a group consisting of: SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6.

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

In one aspect, the invention provides an IB-QX K19-A P162 strain and its descendants, wherein the descendants of the IB-QX K19-A P162 strain are any attenuated descendant IBVs thereof having all of the identifying characteristics of the deposited IB-QX K19-A P162 strain, and wherein the IB-QX K19-A P162 strain is deposited under the deposition number CCTCC NO: V202366.

In one aspect, the invention provides an IB-QX K19-A P171 strain and its descendants, wherein the descendants of the IB-QX K19-A P171 strain are any attenuated descendant IBVs thereof having all of the identifying characteristics of the deposited IB-QX K19-A P171 strain, and wherein the IB-QX K19-A P171 strain is deposited under the deposition number CCTCC NO: V202367.

In one aspect, the invention provides an IB-QX K19-A P179 strain and its descendants, wherein the descendants of the IB-QX K19-A P179 strain are any attenuated descendant IBVs thereof having all of the identifying characteristics of the deposited IB-QX K19-A P179 strain, and wherein the IB-QX K19-A P179 strain is deposited under the deposition number CCTCC NO: V202368.

In one aspect, the invention provides an attenuated infectious bronchitis virus (IBV) , wherein the attenuated IBV is selected from a group consisting of: the IB-QX K19-A P162 strain deposited under the deposition number CCTCC NO: V202366, any one of the descendants of the IB-QX K19-A P162 strain comprising up to 5, 4, 3, 2 or 1 passage of the IB-QX K19-A P162 strain, the IB-QX K19-A P171 strain deposited under the deposition number CCTCC NO: V202367, any one of the descendants of the IB-QX K19-A P171 strain comprising up to 5, 4, 3, 2 or 1 passage of the IB-QX K19-A P171 strain, the IB-QX K19-A P179 strain deposited under the deposition number CCTCC NO: V202368, and any one of the descendants of the IB-QX K19-A P179 strain comprising up to 5, 4, 3, 2 or 1 passage of the IB-QX K19-A P179 strain.

In one aspect, the invention provides a method of preparing an attenuated IBV, comprising a step of: passaging an IBV strain for within 10 passages on a chicken embryo or chicken embryo derived cells; more preferably, passaging an IBV strain for 5, 4, 3, 2 or 1 passage on a chicken embryo or chicken embryo derived cells, wherein the IBV strain is selected from a group consisting of the IB-QX K19-A P162 strain deposited under the deposition number CCTCC NO: V202366, the IB-QX K19-A P171 strain deposited under the deposition number CCTCC NO: V202367, and the IB-QX K19-A P179 strain deposited under the deposition number CCTCC NO: V202368.

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

In one aspect, the invention provides a method of immunizing birds, or a method of preventing or preventing clinical signs caused by IBV in birds, or a method of reducing the ciliostasis in birds, wherein the method comprises administering to the subject a therapeutically or prophylactically effective amount of the immunogenic composition or an attenuated live vaccine of the invention, or a therapeutically or prophylactically effective amount of the immunogenic composition or an attenuated live vaccine prepared by the method of the invention.

In one aspect, the invention provides use of the attenuated IBV of the invention in preparation of an immunogenic composition or an attenuated live vaccine for immunizing birds, for preventing or preventing clinical signs caused by IBV in birds, or for reducing the ciliostasis in birds.

The attenuated IBV strains of the invention were all proven to be safe to chickens and have good genetic stability, without reversion of virulence after several passages. Moreover, the attenuated IBV strains of the invention showed superior protection efficacy against IBV infection, either via oculonasal drop or via spray administration, thereby achieving convenient and large-scale vaccination in a poultry farm. In addition, the attenuated IBV strains of the invention showed superior immunogenicity in a relatively broad range of passages, thereby ensuring more passages can be used for industrial production of an IBV vaccine.

Brief Description of the Drawings

Figure 1: Sequence comparison result of the S1 genes between the IB-QX K19-A P162 strain and the IB-QX K19-A P171 strain.

Figure 2: Sequence comparison result of the S1 genes between the IB-QX K19-A P162 strain and the IB-QX K19-A P179 strain.

Figure 3: Sequence comparison result of the S1 genes between the IB-QX K19-A P171 strain and the IB-QX K19-A P179 strain.

Figure 4: Sequence comparison result of the S1 proteins between the IB-QX K19-A P162 strain and the IB-QX K19-A P171 strain.

Figure 5: Sequence comparison result of the S1 proteins between the IB-QX K19-A P162 strain and the IB-QX K19-A P179 strain.

Figure 6: Sequence comparison result of the S1 proteins between the IB-QX K19-A P171 strain and the IB-QX K19-A P179 strain.

Detailed Description of the Invention

Disclosed are an attenuated IBV, a method of preparing an attenuated IBV, an immunogenic composition or an attenuated live vaccine against an IBV, a method of preparing an immunogenic composition or an attenuated live vaccine against an IBV, and a method of treating or preventing clinical signs caused by IBV in birds.

Before the aspects of the invention are described, it must be noted that as used herein and in the appended claims, the singular forms “a” , “an” , and “the” include plural reference unless the context clearly dictates otherwise. Thus, for example, reference to the "virus" is a reference to one or more viruses and equivalents thereof known to those skilled in the art, and so forth.

The term “and/or” is intended to encompass any combinations of the items connected by this term, equivalent to listing all the combinations individually. For example, “A, B and/or C” encompasses “A” , “B” , “C” , “A and B” , “A and C” , “B and C” , and “A and B and C” .

The terms “include” and “including” have the same meaning as the terms “comprise” and “comprising” in that these latter terms are “open” transitional terms that do not limit claims only to the recited elements succeeding these transitional terms. The term “consisting of, ” while encompassed by the term “comprising, ” should be interpreted as a “closed” transitional term that limits claims only to the recited elements succeeding this transitional term.

Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art in the technical field to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the invention, the preferred methods, devices, and materials are now described. All publications mentioned herein are incorporated herein by reference for the purpose of describing and disclosing the virus strains, the cell lines, vectors, and methodologies as reported in the publications which might be used in connection with the invention. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

Attenuated Infectious Bronchitis Virus (attenuated IBV)

The invention provides an attenuated infectious bronchitis virus (IBV) .

The term “infectious bronchitis virus (IBV) ” refers to an avian bronchitis virus, which belongs to the prototype gammacoronavirus of the family Coronaviridae, order Nidovirales. IBV is an enveloped virus with a positive sense single-stranded RNA genome of 27.6 kb. The viral genome encodes structural proteins, including the spike protein (S) , the envelope protein (E) , the membrane protein (M) and the nucleocapsid protein (N) . Proteins S, E and M are part of the viral envelope while protein N forms the ribonucleoprotein core along with the viral RNA. Protein S is a dimeric ortrimeric transmembrane protein, which is proteolytically cleaved into two subunits, S1 protein and S2 protein. It has been widely known and accepted in the prior arts that the S1 protein is responsible for cell attachment and is the most important antigenic determinant for IBV. S1 gene can also be used for the identification of an IBV strain together with its serotype. As used herein, the term “S1 gene” refers to a polynucleotide encoding the S1 subunit of IBV Protein S.

IBV principally infects the upper respiratory epithelium of chickens among other avian species, causing a respiratory disease. As used herein, IBV is described as a virulent IBV strain unless stated otherwise. The terms “infectious bronchitis virus” , “IBV” and “IBV strain” are exchangeable in the present invention.

IBV strains can be classified by different serotypes through serological studies and/or genetical and phylogenetic analysis of its S1 gene, and serotype QX is one of them. Hence, the term “IB-QX” refers to an IBV of serotype QX. It is known in the art how to determine the serotype of an IBV strain. For example, the serotype of the IBV strain of the invention can be determined by comparing its S1 gene with the published S1 genes whose corresponding serotypes have already been determined.

In the invention, a K19 strain was isolated from the kidney tissue of a chicken with a suspected IBV infection, and then it was identified as a virulent IB-QX strain. As used herein, the term “IB-QX K19 strain” refers to this virulent IB-QX strain. The term “IB-QX K19 P7 strain” refers to another virulent strain which is obtained by passaging the IB-QX K19 strain to its 7^th passage. The IB-QX K19 P7 strain was deposited under the deposition number CCTCC NO: V202364. The term “IB-QX K19 P8 strain” refers to another virulent strain which is obtained by passaging the IB-QX K19 strain to its 8^th passage. The IB-QX K19 P8 strain was deposited under the deposition number CCTCC NO: V202365.

The term “attenuated IBV” refers to an IBV having a reduced virulence in comparison to a virulent IBV strain. In the present invention, an attenuated IBV is one in which the virulence has been reduced so that it does not cause clinical signs of an IBV infection but is capable of inducing an immune response in birds. The term “reduce/reduced” means a virulence reduction of at least 50%, preferably 60%, more preferably 70%, still more preferably 80%, still more preferably 90%, even more preferably 95%and most preferably of 100%as compared to a virulent IBV strain.

Attenuation of a virulent IBV strain can be achieved by passaging the virulent IBV strain at least enough times so that the resulting IBV strain is characterized as having reduce/reduced virulence. In the invention, IB-QX K19 strain was isolated and then was passaged in a chicken embryo to obtain several attenuated IBV strains with different passages. A method of generating an attenuated IBV strain by passaging has been known in the art. An exemplary method of passaging a virulent IBV strain involves the use of a chicken embryo as the environment conducive to virus replication. The chicken embryo is inoculated with a quantity of a virulent IBV strain via the allantoic cavity. The chicken embryo is incubated under suitable conditions, and then the allantoic fluid is harvested from the embryo. At this point, the IBV strain has been passaged one passage (the 1^st passage) . The harvested allantoic fluid from the first passage, at appropriate dilution, is then inoculated into a new chicken embryo, which is incubated again, and then the allantoic fluid is harvested from this second chicken embryo. At this point, the IBV strain has been passaged two passages (the 2^nd passage) . The degree of virulence of an IBV strain can be tested after each passage. Continued passaging in this manner until an attenuated IBV strain is obtained. An exemplary method of attenuating a virulent IBV strain via continuously passaging is illustrated in Example 1.

As used herein, the IB-QX K19 strain is used as a starting point for designating the passage of a passaged IB-QX strain unless stated otherwise. For example, the term “IB-QX K19-A P162 strain” refers to the 162^nd passage of the IB-QX K19 strain. Hence, for example, the expression of “passaging the IB-QX K19 P7 strain to the 162^nd passage” used herein means that the passaging starts from the IB-QX K19 P7 strain and continues until to the 162^nd passage of the IB-QX K19 strain. Thus, unless stated, all of passage numbers described in the invention are determined on the basis of the IB-QX K19 strain. The IB-QX K19-A P162 strain is an attenuated IBV strain, and thus the name of “IB-QX K19-A P162 strain” comprises a symbol “-A” . IB-QX K19-A P162 strain was deposited under the deposition number CCTCC NO: V202366. The nucleotide sequence of the S1 gene of the IB-QX K19-A P162 strain is set forth in SEQ ID NO: 1; and the amino acid sequence encoded by the S1 gene (S1 protein) of the IB-QX K19-A P162 strain is set forth in SEQ ID NO: 4. The detailed method of preparing the IB-QX K19-A P162 strain is illustrated in Example 1.

The term “IB-QX K19-A P171 strain” refers to the 171^st passage of the IB-QX K19 strain. The IB-QX K19-A P171 strain is also an attenuated IBV strain, and it was deposited under the deposition number CCTCC NO: V202367. The nucleotide sequence of the S1 gene of the IB-QX K19-A P171 strain is set forth in SEQ ID NO: 2; and the amino acid sequence encoded by the S1 gene (S1 protein) of the IB-QX K19-A P162 strain is set forth in SEQ ID NO: 5. The detailed method of preparing the IB-QX K19-A P171 strain can be seen from Example 1. The term “IB-QX K19-A P171 strain” has the similar meanings as “IB-QX K19-A P162 strain” .

The term “IB-QX K19-A P179 strain” refers to the 179^th passage of the IB-QX K19 strain. The IB-QX K19-A P179 strain is also an attenuated IBV strain, and it was deposited under the deposition number CCTCC NO: V202368. The nucleotide sequence of the S1 gene of the IB-QX K19-A P179 strain is set forth in SEQ ID NO: 3; and the amino acid sequence encoded by the S1 gene (S1 protein) of the IB-QX K19-A P162 strain is set forth in SEQ ID NO: 6. The detailed method of preparing the IB-QX K19-A P179 strain can be seen from Example 1. The term “IB-QX K19-A P179 strain” has the similar meanings as “IB-QX K19-A P162 strain” .

In one aspect, the present invention provides an attenuated IBV wherein the attenuated IBV comprises a S1 gene comprising or consisting of a nucleotide sequence encoding an amino acid sequence having at least 95%sequence identity to the amino acid sequence set forth in SEQ ID NO: 4.

The term “sequence identity” refers to a relationship between two or more amino acid sequences or two or more nucleic acid sequences, namely a reference sequence and a given sequence to be compared with the reference sequence. Sequence identity is determined by comparing the given sequence to the reference sequence after the sequences have been optimally aligned to produce the highest degree of sequence similarity, as determined by the match between strings of such sequences. A method of calculating the sequence identity between two or more amino acid/nucleic acid sequences is well-known in the art.

In some embodiments, the attenuated IBV comprises a S1 gene comprising or consisting of a nucleotide sequence encoding an amino acid sequence having at least 96%, 97%, 98%, 99%or 100%sequence identity to the amino acid sequence set forth in SEQ ID NO: 4. In some embodiments, the attenuated IBV comprises a S1 gene comprising or consisting 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 to the amino acid sequence set forth in SEQ ID NO: 4. In some embodiments, the attenuated IBV comprises a S1 gene comprising or consisting of a nucleotide sequence encoding an amino acid sequence selected from a group consisting of: SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6.

In some embodiments, the attenuated IBV comprises a S1 gene comprising or consisting of a nucleotide sequence having at least 95%sequence identity to the nucleotide sequence set forth in SEQ ID NO: 1. In some embodiments, the attenuated IBV comprises a S1 gene comprising or consisting of a nucleotide sequence having at least 96%, 97%, 98%, 99%or 100%sequence identity to the nucleotide sequence set forth in SEQ ID NO: 1. In some embodiments, the attenuated IBV comprises a S1 gene comprising or consisting 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 to the nucleotide sequence set forth in SEQ ID NO: 1. In some embodiments, the attenuated IBV comprises a S1 gene comprising or consisting 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 to the nucleotide sequence set forth in SEQ ID NO: 1. In some embodiments, the attenuated IBV comprises a S1 gene comprising or consisting of a nucleotide sequence having at least 99.9%or 100%sequence identity to the nucleotide sequence set forth in SEQ ID NO: 1. In some embodiments, the S1 gene of the attenuated IBV comprises or consists of a nucleotide sequence selected from a group consisting of: SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3.

In some embodiments, the attenuated IBV is of a QX serotype. In some embodiments, the attenuated IBV is derived from the IB-QX K19 P7 strain.

In some embodiments, the attenuated IBV is selected from a group consisting of: the IB-QX K19-A P162 strain, the IB-QX K19-A P171 strain, and the IB-QX K19-A P179 strain. In some embodiments, the attenuated IBV is isolated.

It was shown in the examples that the S1 genes/proteins of the IB-QX K19-A P162 strain, the IB-QX K19-A P171 strain and the IB-QX K19-A P179 strain nearly keep the same (more than 99.8%sequence identity, see Figs. 1-6) , and they are all safe to chickens and could protect chickens against IBV infection.

In another aspect, the present invention provides an attenuated IBV, wherein the attenuated IBV is obtainable by passaging the IB-QX K19 P7 strain deposited under the deposition number CCTCC NO: V202364 to at least the 160^th passage.

In some embodiments, the attenuated IBV is obtainable by passaging the IB-QX K19 P7 strain to the 160^th to 200^th passages. In some embodiments, the attenuated IBV is obtainable by passaging the IB-QX K19 P7 strain to the 160^th to 180^th passages. In some embodiments, the attenuated IBV is obtainable by passaging the IB-QX K19 P7 strain to the 162^nd to 179^th passages. In some embodiments, the attenuated IBV is obtainable by passaging the IB-QX K19 P7 strain to the 162^nd, 163^rd, 164^th, 165^th, 166^th, 167^th, 168^th, 169^th, 170^th, 171^st, 172^nd, 173^rd, 174^th, 175^th, 176^th, 177^th, 178^th or 179^th passage. In some embodiments, the attenuated IBV is obtainable by passaging the IB-QX K19 P7 strain to the 162^nd, 171^st or 179^th passage. The term “obtainable” is used herein for defining the attenuated IBV by its preparation process. For the purposes of the invention, the term “obtainable” encompasses the term “obtained” .

In some embodiments, the attenuated IBV comprises a S1 gene comprising or consisting of a nucleotide sequence encoding an amino acid sequence having at least 95%sequence identity to the amino acid sequence set forth in SEQ ID NO: 4. In some embodiments, the attenuated IBV comprises a S1 gene comprising or consisting of a nucleotide sequence encoding an amino acid sequence having at least 96%, 97%, 98%, 99%or 100%sequence identity to the amino acid sequence set forth in SEQ ID NO: 4. In some embodiments, the attenuated IBV comprises a S1 gene comprising or consisting 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 to the amino acid sequence set forth in SEQ ID NO: 4. In some embodiments, the attenuated IBV comprises a S1 gene comprising or consisting of a nucleotide sequence encoding an amino acid sequence selected from a group consisting of: SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6.

In some embodiments, the attenuated IBV is obtainable by passaging the IB-QX K19 P7 strain to the 162^nd passage, and the S1 gene of the attenuated IBV comprises or consists of a nucleotide sequence encoding an amino acid sequence selected from a group consisting of: SEQ ID NO: 4, preferably the S1 gene of the attenuated IBV comprises or consists of a nucleotide sequence set forth in SEQ ID NO: 1.

In some embodiments, the attenuated IBV is obtainable by passaging the IB-QX K19 P7 strain to the 171^st passage, and the S1 gene of the attenuated IBV comprises or consists of a nucleotide sequence encoding an amino acid sequence selected from a group consisting of: SEQ ID NO: 5, preferably the S1 gene of the attenuated IBV comprises or consists of a nucleotide sequence set forth in SEQ ID NO: 2.

In some embodiments, the attenuated IBV is obtainable by passaging the IB-QX K19 P7 strain to the 179^th passage, and the S1 gene of the attenuated IBV comprises or consists of a nucleotide sequence encoding an amino acid sequence selected from a group consisting of: SEQ ID NO: 6, preferably the S1 gene of the attenuated IBV comprises or consists of a nucleotide sequence set forth in SEQ ID NO: 3.

In another aspect, the present invention provides an attenuated infectious bronchitis virus (IBV) , wherein the attenuated IBV is selected from a group consisting of: the IB-QX K19-A P162 strain deposited under the deposition number CCTCC NO: V202366, the IB-QX K19-A P171 strain deposited under the deposition number CCTCC NO: V202367, and the IB-QX K19-A P179 strain deposited under the deposition number CCTCC NO: V202368.

In some embodiments, the attenuated IBV is isolated. As used herein, the term “isolated” means that the attenuated IBVs are not contained within a tissue of a live animal.

In another aspect, the present invention further provides a method of preparing an attenuated IBV, comprising a step of passaging the IB-QX K19 P7 strain deposited under the deposition number CCTCC NO: V202364 to at least the 160th passage on a chicken embryo or chicken embryo derived cells.

In some embodiments, the method comprises a step of passaging the IB-QX K19 P7 strain to the 160^th to 200^th passages on a chicken embryo or chicken embryo derived cells. In some embodiments, the method comprises a step of passaging the IB-QX K19 P7 strain to the 160^th to 180^th passages on a chicken embryo or chicken embryo derived cells. In some embodiments, the method comprises a step of passaging the IB-QX K19 P7 strain to the 162^nd to 179^th passages on a chicken embryo or chicken embryo derived cells. In some embodiments, the method comprises a step of passaging the IB-QX K19 P7 strain to the 162^nd, 163^rd, 164^th, 165^th, 166^th, 167^th, 168^th, 169^th, 170^th, 171^st, 172^nd, 173^rd, 174^th, 175^th, 176^th, 177^th, 178^th or 179^th passage on a chicken embryo or chicken embryo derived cells. In some embodiments, the method comprises a step of passaging the IB-QX K19 P7 strain to the 162nd, 171^st or 179^th passage on a chicken embryo or chicken embryo derived cells.

In some embodiments, the method further comprises a step of harvesting allantoic fluid of the chicken embryo or the cell culture and filtering to obtain the attenuated IBV strain of the invention. 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 the invention, an IB-QX K19 strain was used as a parent strain for producing several attenuated IBV strains. The produced attenuated IBV strains were all safe to chickens and occurred no reversion of virulence. Moreover, the produced attenuated IBV strains showed superior protection efficacy against IBV infection in chickens, with a protection rate higher than 90%; amongst the IB-QX K19-A P162 strain still shows very strong protection efficacy even at a dose amount as low as 10^3.0EID₅₀ per chicken.

Descendant of attenuated IBV

The present invention also provides any descendants of the deposited attenuated IBV strains of the invention.

As used herein, when an IBV strain X is passaged from an IBV strain Y, the IBV strain X can be called as the descendant of the IBV strain Y. For example, a descendant of the IB-QX K19-A P162 strain can be obtained by inoculating a quantity of the IB-QX K19-A P162 strain into an allantoic cavity of a chicken embryo, incubating the chicken embryo under suitable conditions (such as 37℃ for 24 h) , harvesting allantoic fluid from the chicken embryo and isolating a virus strain. The isolated virus strain is a descendant of the IB-QX K19-A P162 strain. The above process can be repeated several times, thereby obtaining several descendants of the IB-QX K19-A P162 strain with different passages.

The descendants of the deposited attenuated IBV strains of the invention are also attenuated ones, and have all of the identifying characteristics of the deposited attenuated IBV strains. “The identifying characteristics of the deposited attenuated IBV strains” comprise but are not limited to the S1 gene/protein sequence of the deposited attenuated IBV strains, the protection properties of the deposited attenuated IBV strains against virulent IB-QX challenge, etc.

In some embodiments, the identifying characteristic of the deposited attenuated IBV strain is the S1 protein sequence of the deposited attenuated IBV strain, or a S1 protein having at least 99%, preferable at least 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%or 100%sequence identity to the S1 protein of the deposited attenuated IBV strain. In some embodiments, the identifying characteristic of the deposited attenuated IBV strain is the S1 gene sequence of the deposited attenuated IBV strain, or a S1 gene having at least 99%, preferable at least 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%or 100%sequence identity to the S1 gene of the deposited attenuated IBV strain.

In some embodiments, the identifying characteristic of the deposited attenuated IBV strains is the protection property which can provide a protection against virulent IB-QX challenge in at least 80%, preferably 85%, more preferably 90%, more preferably 95%, and most preferably 100%of birds. In some embodiments, the identifying characteristic of the deposited attenuated IBV strains is the protection property which can provide an improved ciliostasis score after virulent IB-QX challenge in at least 90%, preferably 95%, and most preferably 100%of birds. In the invention, the term “attenuated IBV” also encompasses any one of the descendants of an attenuated IBV.

In some embodiments, the descendants are the attenuated descendants of the IB-QX K19-A P162 strain having all of the identifying characteristics of the IB-QX K19-A P162 strain. In some embodiments, the attenuated descendants of the IB-QX K19-A P162 strain comprise the same S1 protein as that of the IB-QX K19-A P162 strain set forth in SEQ ID NO: 4, or a S1 protein having at least 99%, preferable at least 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%or 100%sequence identity to the S1 protein of the IB-QX K19-A P162 strain set forth in SEQ ID NO: 4. In some embodiments, the attenuated descendants of the IB-QX K19-A P162 strain comprise the same S1 gene (SEQ ID NO: 1) as that of the IB-QX K19-A P162 strain, or a S1 gene having at least 99%, preferable at least 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%or 100%sequence identity to the S1 of the IB-QX K19-A P162 strain. In some embodiments, the attenuated descendants of the IB-QX K19-A P162 strain can protect birds against virulent IB-QX challenge, including the protection against virulent IB-QX challenge in at least 80%, preferably 85%, more preferably 90%, more preferably 95%, and most preferably 100%of birds, or the improved ciliostasis score after virulent IB-QX challenge in at least 90%, preferably 95%, and most preferably 100%of birds.

In some embodiments, the descendants are the attenuated descendants of the IB-QX K19-A P171 strain having all of the identifying characteristics of the IB-QX K19-A P171 strain. In some embodiments, the attenuated descendants of the IB-QX K19-A P171 strain comprise the same S1 protein as that of the IB-QX K19-A P171 strain set forth in SEQ ID NO: 5, or a S1 protein having at least 99%, preferable at least 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%or 100%sequence identity to the S1 protein of the IB-QX K19-A P171 strain set forth in SEQ ID NO: 5. In some embodiments, the attenuated descendants of the IB-QX K19-A P171 strain comprise the same S1 gene (SEQ ID NO: 2) as that of the IB-QX K19-A P171 strain, or a S1 gene having at least 99%, preferable at least 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%or 100%sequence identity to the S1 of the IB-QX K19-A P171 strain. In some embodiments, the attenuated descendants of the IB-QX K19-A P171 strain can protect birds against virulent IB-QX challenge, including the protection against virulent IB-QX challenge in at least 80%, preferably 85%, more preferably 90%, more preferably 95%, and most preferably 100%of birds, or the improved ciliostasis score after virulent IB-QX challenge in at least 90%, preferably 95%, and most preferably 100%of birds.

In some embodiments, the descendants are the attenuated descendants of the IB-QX K19-A P179 strain having all of the identifying characteristics of the IB-QX K19-A P179 strain. In some embodiments, the attenuated descendants of the IB-QX K19-A P179 strain comprise the same S1 protein as that of the IB-QX K19-A P179 strain set forth in SEQ ID NO: 6, or a S1 protein having at least 99%, preferable at least 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%or 100%sequence identity to the S1 protein of the IB-QX K19-A P179 strain set forth in SEQ ID NO: 6. In some embodiments, the attenuated descendants of the IB-QX K19-A P179 strain comprise the same S1 gene (SEQ ID NO: 3) as that of the IB-QX K19-A P179 strain, or a S1 gene having at least 99%, preferable at least 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%or 100%sequence identity to the S1 of the IB-QX K19-A P179 strain. In some embodiments, the attenuated descendants of the IB-QX K19-A P179 strain can protect birds against virulent IB-QX challenge, including the protection against virulent IB-QX challenge in at least 80%, preferably 85%, more preferably 90%, more preferably 95%, and most preferably 100%of birds, or the improved ciliostasis score after virulent IB-QX challenge in at least 90%, preferably 95%, and most preferably 100%of birds.

In some embodiments, the descendants comprise up to 10 passages of the deposited attenuated IBV strains of the invention. In some embodiments, the descendants comprise up to 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 passage of the deposited attenuated IBV strains of the invention.

In some embodiments, the descendants comprise up to 10 passages of the IB-QX K19-A P162 strain. In some embodiments, the descendants comprise up to 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 passage of the IB-QX K19-A P162 strain. In some embodiments, the descendant is the 10^th, 9^th, 8^th, 7^th, 6^th, 5^th, 4^th, 3^rd, 2^nd or 1^st passage of the IB-QX K19-A P162 strain. In some embodiments, the descendant is the 5^th, 4^th, 3^rd, 2^nd or 1^st passage of the IB-QX K19-A P162 strain. In some embodiments, the descendant is obtainable by passaging the IB-QX K19-A P162 strain for 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 passage. In some embodiments, the descendant is obtainable by passaging the IB-QX K19-A P162 strain for 5, 4, 3, 2 or 1 passage.

In some embodiments, the descendants comprise up to 10 passages of the IB-QX K19-A P171 strain. In some embodiments, the descendants comprise up to 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 passage of the IB-QX K19-A P171 strain. In some embodiments, the descendant is the 10^th, 9^th, 8^th, 7^th, 6^th, 5^th, 4^th, 3^rd, 2^nd or 1^st passage of the IB-QX K19-A P171 strain. In some embodiments, the descendant is the 5^th, 4^th, 3^rd, 2^nd or 1^st passage of the IB-QX K19-A P171 strain. In some embodiments, the descendant is obtainable by passaging the IB-QX K19-A P171 strain for 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 passage. In some embodiments, the descendant is obtainable by passaging the IB-QX K19-A P171 strain for 5, 4, 3, 2 or 1 passage.

In some embodiments, the descendants comprise up to 10 passages of the IB-QX K19-A P179 strain. In some embodiments, the descendants comprise up to 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 passage of the IB-QX K19-A P179 strain. In some embodiments, the descendant is the 10^th, 9^th, 8^th, 7^th, 6^th, 5^th, 4^th, 3^rd, 2^nd or 1^st passage of the IB-QX K19-A P179 strain. In some embodiments, the descendant is the 5^th, 4^th, 3^rd, 2^nd or 1^st passage of the IB-QX K19-A P179 strain. In some embodiments, the descendant is obtainable by passaging the IB-QX K19-A P179 strain for 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 passage. In some embodiments, the descendant is obtainable by passaging the IB-QX K19-A P179 strain for 5, 4, 3, 2 or 1 passage.

In another aspect, the present invention provides an attenuated infectious bronchitis virus (IBV) , wherein the attenuated IBV is selected from a group consisting of: the IB-QX K19-A P162 strain deposited under the deposition number CCTCC NO: V202366, any one of the descendants of the IB-QX K19-A P162 strain comprising up to 5, 4, 3, 2 or 1 passage of the IB-QX K19-A P162 strain (for example, the 5^th, 4^th, 3^rd, 2^nd or 1^st passage of the IB-QX K19-A P162 strain) , the IB-QX K19-A P171 strain deposited under the deposition number CCTCC NO: V202367, any one of the descendants of the IB-QX K19-A P171 strain comprising up to 5, 4, 3, 2 or 1 passage of the IB-QX K19-A P171 strain (for example, the 5^th, 4^th, 3^rd, 2^nd or 1^st passage of the IB-QX K19-A P171 strain) , the IB-QX K19-A P179 strain deposited under the deposition number CCTCC NO: V202368, and any one of the descendants of the IB-QX K19-A P179 strain comprising up to 5, 4, 3, 2 or 1 passage of the IB-QX K19-A P179 strain (for example, the 5^th, 4^th, 3^rd, 2^nd or 1^st passage of the IB-QX K19-A P179 strain) .

In another aspect, the present invention provides a method of preparing any descendants of the deposited attenuated IBV strains of the invention.

In some embodiments, the method comprises a step of passaging the IB-QX K19-A P162 strain deposited under the deposition number CCTCC NO: V202366 for within 10 passages on a chicken embryo or chicken embryo derived cells. In some embodiments, the method comprises a step of passaging the IB-QX K19-A P162 strain deposited under the deposition number CCTCC NO: V202366 for 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 passage on a chicken embryo or chicken embryo derived cells. In some embodiments, the method comprises a step of passaging the IB-QX K19-A P162 strain deposited under the deposition number CCTCC NO: V202366 for 5, 4, 3, 2 or 1 passage on a chicken embryo or chicken embryo derived cells.

In some embodiments, the method comprises a step of passaging the IB-QX K19-A P171 strain deposited under the deposition number CCTCC NO: V202367 for within 10 passages on a chicken embryo or chicken embryo derived cells. In some embodiments, the method comprises a step of passaging the IB-QX K19-A P171 strain deposited under the deposition number CCTCC NO: V202367 for 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 passage on a chicken embryo or chicken embryo derived cells. In some embodiments, the method comprises a step of passaging the IB-QX K19-A P171 strain deposited under the deposition number CCTCC NO: V202367 for 5, 4, 3, 2 or 1 passage on a chicken embryo or chicken embryo derived cells.

In some embodiments, the method comprises a step of passaging the IB-QX K19-A P179 strain deposited under the deposition number CCTCC NO: V202368 for within 10 passages on a chicken embryo or chicken embryo derived cells. In some embodiments, the method comprises a step of passaging the IB-QX K19-A P179 strain deposited under the deposition number CCTCC NO: V202368 for 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 passage on a chicken embryo or chicken embryo derived cells. In some embodiments, the method comprises a step of passaging the IB-QX K19-A P179 strain deposited under the deposition number CCTCC NO: V202368 for 5, 4, 3, 2 or 1 passage on a chicken embryo or chicken embryo derived cells.

In some embodiments, the method further comprises a step of harvesting allantoic fluid of the chicken embryo or the cell culture and filtering to obtain a descendant of the deposited attenuated IBV strain of the invention.

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.

It was surprisingly found in the examples that the descendants of the deposited attenuated IBV strains could still keep the same immunological properties as the deposited attenuated IBV strains, and the deposited attenuated IBV strains and their descendants within at least 10 passages showed superior immune protection efficacy against virulent IB-QX challenge in chickens.

Immunogenic composition and vaccine

The present invention provides an immunogenic composition or an attenuated live vaccine against an infection of an IBV.

The term “immunogenic composition” refers to a composition that comprises at least one antigen, which elicits an immunological response in the host to which the immunogenic composition is administered. As used herein, the immunogenic composition confers protective immunity against one or more of the clinical signs of an IBV infection. In case where the host displays a protective immunological response such that resistance to new infection will be enhanced and/or the clinical severity of the disease reduced, the immunogenic composition is described as a “vaccine” .

In one aspect, the present invention provides an immunogenic composition or an attenuated live vaccine against an infection of an IBV.

In some embodiments, the immunogenic composition or the attenuated live vaccine comprises a therapeutically or prophylactically effective amount of the attenuated IBV of the invention, or a therapeutically or prophylactically effective amount of the attenuated IBV prepared by the method of the invention.

The term “an effective amount” refers to an amount of antigen that elicits or is able to elicit an immune response in birds. Such effective amount is able to lessen the incidence of the particular IBV infection in birds or to reduce the severity of clinical signs of the particular IBV infection. In some embodiments, clinical signs are lessened in incidence or severity by at least 10%, more preferably by at least 20%, still more preferably by at least 30%, even more preferably by at least 40%, still more preferably by at least 50%, even more preferably by at least 60%, still more preferably by at least 70%, even more preferably by at least 80%, still more preferably by at least 90%, still more preferably by at least 95%and most preferably by 100%in comparison to birds that are not treated/prevented with an attenuated IBV, an immunogenic composition or a vaccine of the invention.

In some embodiments, the immunogenic composition or the attenuated live vaccine comprises an amount of 1 to 10 log₁₀ EID₅₀ per ml of the attenuated IBV, including 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 log₁₀ EID₅₀ per ml of the attenuated IBV. In some embodiments, the immunogenic composition or the attenuated live vaccine comprises an amount of 2 to 8 log₁₀ EID₅₀ per ml of the attenuated IBV. In some embodiments, the immunogenic composition or the attenuated live vaccine comprises an amount of 3 to 8 log₁₀ EID₅₀ per ml of the attenuated IBV. In some embodiments, the immunogenic composition or the attenuated live vaccine comprises an amount of 5 to 8 log₁₀ EID₅₀ per ml of the attenuated IBV. In some embodiments, the immunogenic composition or the attenuated live vaccine comprises an amount of 6 to 7 log₁₀ EID₅₀ per ml of the attenuated IBV. In some embodiments, the immunogenic composition or the attenuated live vaccine comprises an amount of 6.5 log₁₀ EID₅₀ per ml of the attenuated IBV.

In some embodiments, the immunogenic composition or the attenuated live vaccine comprises at least a veterinary acceptable carrier. In some embodiments, the veterinary acceptable carrier is a lyoprotectant.

In some embodiments, the immunogenic composition or the attenuated live vaccine is formulated for a single dose administration. In some embodiments, the immunogenic composition or the attenuated live vaccine is administered by an eye drop, a nasal drop, or an oculonasal drop. In some embodiments, the immunogenic composition or the attenuated live vaccine is administered via spray.

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

In some embodiments, the method comprises steps of:

(1) preparing the attenuated IBV of the invention;

(2) adding a veterinary acceptable carrier such as a lyoprotectant, mixing to obtain the immunogenic composition or an attenuated live vaccine against IBV infection.

The attenuated IBV of the invention has been described in the above contents. An exemplary method of preparing an attenuated IBV of the invention has also been described in the above contents.

In some embodiments, a method of preparing an attenuated IBV of the invention comprises a step of: passaging the IB-QX K19 P7 strain deposited under the deposition number CCTCC NO: V202364 to at least the 160^th passage on a chicken embryo or chicken embryo derived cells; preferably, passaging the IB-QX K19 P7 strain to the 160^th to 200^th passages on a chicken embryo or chicken embryo derived cells; more preferably, passaging the IB-QX K19 P7 strain to the 160^th to 180^th passages on a chicken embryo or chicken embryo derived cells; more preferably, passaging the IB-QX K19 P7 strain to the 162^nd to 179^th passages on a chicken embryo or chicken embryo derived cells; more preferably, passaging the IB-QX K19 P7 strain to the 162^nd, 163^rd, 164^th, 165^th, 166^th, 167^th, 168^th, 169^th, 170^th, 171^st, 172^nd, 173^rd, 174^th, 175^th, 176^th, 177^th, 178^th or 179^th passage on a chicken embryo or chicken embryo derived cells; most preferably, passaging the IB-QX K19 P7 strain to the 162^nd, 171^st or 179^th passage on a chicken embryo or chicken embryo derived cells. The methods of preparing attenuated IBV of the invention are the same as those described in the above contents.

The attenuated IBV strains of the invention showed superior immunogenicity in a wide range of 20 passages, and therefore they are very practically for industrial production of an immunogenic composition or an attenuated live IBV vaccine.

Treatment and prevention method

The present invention provides a method for immunizing birds, or a method of treating or preventing clinical signs caused by IBV in birds, or a method of reducing the ciliostasis in birds.

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

In one aspect, the present invention provides an immunogenic composition or a vaccine of the invention for use in a method of immunizing birds, comprising a step of administering to such birds a therapeutically or prophylactically effective amount of an attenuated IBV, an immunogenic composition or a vaccine of the invention.

In one aspect, the present invention provides use of the attenuated IBV of the invention in preparation of an immunogenic composition or a vaccine of the invention for immunizing birds.

The term “immunizing” relates to an active immunization by the administration of an attenuated IBV, an immunogenic composition or a vaccine of the invention to birds to be immunized, thereby causing an immunological response against the antigen included in such an attenuated IBV, immunogenic composition or a vaccine of the invention. Immunization results in lessening of the incidence of the particular IBV infection in birds or in the reduction in the severity of clinical signs caused by or associated with the particular IBV infection.

It is to be understood that immunization may not be effective in each immunized individual of birds. However, the term requires that a significant portion of birds are effectively immunized. In some embodiments, the immunization shall be effective if clinical signs in at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, still more preferably in at least 95%and most preferably in 100%of the birds to be administered are lessened in incidence or severity by at least at least 50%, more preferably by at least 60%, still more preferably by at least 70%, even more preferably by at least 80%, still more preferably by at least 90%, still more preferably by at least 95%and most preferably by 100%in comparison to birds that are not immunized with an attenuated IBV, an immunogenic composition or a vaccine of the invention.

The term “birds” is well known to the person skilled in the art. Throughout the invention, the term “birds” encompasses poultry, including such as chickens, turkeys, quails, pheasants, guineafowl, geese, and ducks. The term “chicken” includes broiler, laying hens, and reproductive stocks for both (also referred as breeders) . In some embodiments, the birds are poultry. In some embodiments, the poultry is selected from a group consisting of chickens, turkeys, quails, pheasants, guineafowl, geese, and ducks. In some embodiments, the poultry is selected from a group consisting of chickens, turkeys, quails, and pheasants. In some embodiments, the chicken is selected from a group consisting of broiler, laying hens, and breeders.

In some embodiments, the birds to be immunized are within the first week of age, within the first three days of age, within the first two days of age, or within the first day of age. In some embodiments, the birds 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 of age. In some embodiments, the birds to be immunized are 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 days of age. In some embodiments, the birds to be immunized are 1, 2, 3, 4, 5, 6 or 7 days of age. In some embodiments, the birds to be immunized are within the first day of age. In some embodiments, the birds are chickens, and the chicken is within the first day of age.

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

In one aspect, the present invention provides an immunogenic composition or a vaccine of the invention for use in a method of treating or preventing clinical signs caused by IBV in birds, comprising a step of administering to the birds a therapeutically or prophylactically effective amount of an attenuated IBV, an immunogenic composition or a vaccine of the invention

In one aspect, the present invention provides use of the attenuated IBV of the invention in preparation of an immunogenic composition or a vaccine of the invention for treating or preventing clinical signs caused by IBV in birds.

The term “treating/treatment” refers to the administration of the effective amount of an attenuated IBV, an immunogenic composition or a vaccine of the invention once birds have already been infected with such IBV and/or birds have already shown some clinical signs caused by or associated with such IBV infection. The term “preventing/prevention” refers to the administration of birds prior to any infection of such birds with IBV or at least such birds do not show any clinical signs caused by or associated with the infection by such IBV.

The term “clinical signs” refers to signs of IBV infection in birds. In some embodiments, the clinical signs caused by IBV infection are selected from a group consisting of: ciliostasis, rales, egg drop, kidney lesions, watery diarrhea, weight loss, virus load, viral shedding and combinations thereof. In some embodiments, the clinical signs caused by IBV are ciliostasis.

In one aspect, the present invention provides a method of reducing the ciliostasis in birds, in comparison to non-immunized birds, comprising a step of administering to the birds a therapeutically or prophylactically effective amount of an attenuated IBV, an immunogenic composition or a vaccine of the invention.

In one aspect, the present invention provides an immunogenic composition or a vaccine of the invention for use in a method of reducing the ciliostasis in birds, in comparison to non-immunized birds, comprising a step of administering to the birds a therapeutically or prophylactically effective amount of an attenuated IBV, an immunogenic composition or a vaccine of the invention.

In one aspect, the present invention provides use of the attenuated IBV of the invention in preparation of an immunogenic composition or a vaccine of the invention for reducing the ciliostasis in birds, in comparison to non-immunized birds.

The term “ciliostasis” is well known to the person skilled in that art. The surface of the trachea is covered with specialized epithelial cells, which are lined with numerous, motile, hair-like structures called cilia. The term “ciliostasis” encompasses the reduction or loss of cilia and/or loss or partial loss of ciliary activity (movement) . Ciliostasis may be determined by examining the inner lining of the tracheal rings for the movement of the cilia. It is in the general knowledge of a person skilled in the art how to determine the movement of the cilia in the trachea.

The term “reduction of ciliostasis /reducing the ciliostasis” means, that the ciliostasis is reduced by at least 10%, preferably by at least 20%, more preferably by at least 30%, even more preferably by at least 40%, even more preferably by at least 50%, even more preferably by at least 60%, even more preferably by at least 70%, even more preferably by at least 80%, even more preferably by at least 90%, even more preferably by at least 95%and most preferably by 100%as compared to non-immunized birds. It is in the general knowledge of a person skilled in the art how to measure the reduction of the ciliostasis, and an exemplary method of evaluating the reduction of the ciliostasis is illustrated in Example 3.

In some embodiments, the immunogenic composition or the vaccine of the invention is formulated for a single dose administration.

In some embodiments, the immunogenic composition or the vaccine of the invention is administered by an eye drop, a nasal drop or an oculonasal drop. In some embodiments, the immunogenic composition or the vaccine of the invention is administered via spray.

In some embodiments, the immunogenic composition or the vaccine of the invention comprises an amount of 1 to 10 log₁₀ EID₅₀ per ml of the attenuated IBV, including 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 log₁₀ EID₅₀ per ml of the attenuated IBV. In some embodiments, the immunogenic composition or the vaccine of the invention comprises an amount of 2 to 8 log₁₀ EID₅₀ per ml of the attenuated IBV. In some embodiments, the immunogenic composition or the attenuated live vaccine of the invention comprises an amount of 3 to 8 log₁₀ EID₅₀ per ml of the attenuated IBV. In some embodiments, the immunogenic composition or the vaccine of the invention comprises an amount of 5 to 8 log₁₀ EID₅₀ per ml of the attenuated IBV. In some embodiments, the immunogenic composition or the vaccine of the invention comprises an amount of 6 to 7 log₁₀ EID₅₀ per ml of the attenuated IBV. In some embodiments, the immunogenic composition or the vaccine of the invention comprises an amount of 6.5 log₁₀ EID₅₀ per ml of the attenuated IBV.

It was shown in the examples that one does administration of the attenuated IBVs of the invention could provide enough protection efficacy on 1-day-old chicken against IBV infection, either via an oculonasal drop or spray.

Kits

The immunogenic composition or the vaccine of the invention may, if desired, be presented in a pack or dispenser device which may contain one or more unit dose forms containing the active antigenic ingredient. The pack may for example comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration preferably for administration to birds, especially birds. Associated with such container (s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for administration.

In one aspect, the present invention provides a kit comprising the attenuated IBV, the immunogenic composition or the vaccine of the invention. In some embodiments, the kit further comprises an instruction letter for the treatment and/or prevention of the infection of IBV of birds. In some embodiments, the kit further comprises a dispenser capable of administering the attenuated IBV, the immunogenic composition or the vaccine of the invention to birds.

The following clauses are also described herein and part of disclosure of the invention:

CLAUSES

CLAUSE SET A

1A. An attenuated infectious bronchitis virus (IBV) , wherein the attenuated IBV comprises a S1 gene comprising or consisting of a nucleotide sequence encoding an amino acid sequence having at least 95%sequence identity to the amino acid sequence set forth in SEQ ID NO: 4;

preferably, wherein the attenuated IBV comprises a S1 gene comprising or consisting of a nucleotide sequence encoding an amino acid sequence having at least 96%, 97%, 98%, 99%or 100%sequence identity to the amino acid sequence set forth in SEQ ID NO: 4;

more preferably, wherein the attenuated IBV comprises a S1 gene comprising or consisting 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 to the amino acid sequence set forth in SEQ ID NO: 4;

or

the attenuated IBV comprises a S1 gene comprising or consisting of a nucleotide sequence encoding an amino acid sequence selected from a group consisting of: SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6.

2A. The attenuated IBV of clause 1A, wherein the S1 gene comprises or consists of a nucleotide sequence having at least 95%sequence identity to the nucleotide sequence set forth in SEQ ID NO: 1;

preferably, wherein the S1 gene comprises or consists of a nucleotide sequence having at least 96%, 97%, 98%, 99%or 100%sequence identity to the nucleotide sequence set forth in SEQ ID NO: 1;

more preferably, wherein the S1 gene comprises or consists 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 to the nucleotide sequence set forth in SEQ ID NO: 1;

or

the S1 gene comprises or consists of a nucleotide sequence selected from a group consisting of: SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3.

3A. The attenuated IBV of clause 1A or 2A, wherein the attenuated IBV is of a QX serotype; preferably wherein the attenuated IBV is derived from the IB-QX K19 P7 strain deposited under the deposition number CCTCC NO: V202364.

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

5A. An attenuated infectious bronchitis virus (IBV) , wherein the attenuated IBV is obtainable by passaging the IB-QX K19 P7 strain deposited under the deposition number CCTCC NO: V202364 to at least the 160^th passage; preferably the 160^th to 200^th passages; more preferably, the 160^th to 180^th passages; more preferably, the 162^nd to 179^th passages; more preferably, the 162^nd, 163^rd, 164^th, 165^th, 166^th, 167^th, 168^th, 169^th, 170^th, 171^st, 172^nd, 173^rd, 174^th, 175^th, 176^th, 177^th, 178^th or 179^th passage; and most preferably, the 162^nd, 171^st or 179^th passage.

6A. The attenuated IBV of clause 5A, wherein the attenuated IBV comprises a S1 gene comprising or consisting of a nucleotide sequence encoding an amino acid sequence having at least 95%sequence identity to the amino acid sequence set forth in SEQ ID NO: 4;

or

7A. The attenuated IBV of clause 5A or 6A, wherein the S1 gene comprises or consists of a nucleotide sequence having at least 95%sequence identity to the nucleotide sequence set forth in SEQ ID NO: 1;

or

8A. An attenuated infectious bronchitis virus (IBV) ,

wherein the attenuated IBV is the IB-QX K19-A P162 strain deposited under the deposition number CCTCC NO: V202366 and its descendants, and the descendants of the IB-QX K19-A P162 strain are any attenuated descendant IBVs thereof having all of the identifying characteristics of the deposited IB-QX K19-A P162 strain, including for example, the same S1 protein as that of the IB-QX K19-A P162 strain set forth in SEQ ID NO: 4, or a S1 protein having at least 99%, preferable at least 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%or 100%sequence identity to the S1 protein of the IB-QX K19-A P162 strain set forth in SEQ ID NO: 4, and/or the protection against virulent IB-QX challenge, including the protection against virulent IB-QX challenge in at least 80%, preferably 85%, more preferably 90%, more preferably 95%, and most preferably 100%of birds, or the improved ciliostasis score after virulent IB-QX challenge in at least 90%, preferably 95%, and most preferably 100%of birds;

wherein the attenuated IBV is the IB-QX K19-A P171 strain deposited under the deposition number CCTCC NO: V202367 and its descendants, and the descendants of the IB-QX K19-A P171 strain are any attenuated descendant IBVs thereof having all of the identifying characteristics of the deposited IB-QX K19-A P171 strain, including for example, the same S1 protein as that of the IB-QX K19-A P171 strain set forth in SEQ ID NO: 5, or a S1 protein having at least 99%, preferable at least 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%or 100%sequence identity to the S1 protein of the IB-QX K19-A P171 strain set forth in SEQ ID NO: 5, and/or the protection against virulent IB-QX challenge, including the protection against virulent IB-QX challenge in at least 80%, preferably 85%, more preferably 90%, more preferably 95%, and most preferably 100%of birds, or the improved ciliostasis score after virulent IB-QX challenge in at least 90%, preferably 95%, and most preferably 100%of birds; or

wherein the attenuated IBV is the IB-QX K19-A P179 strain deposited under the deposition number CCTCC NO: V202368 and its descendants, and the descendants of the IB-QX K19-A P179 strain are any attenuated descendant IBVs thereof having all of the identifying characteristics of the deposited IB-QX K19-A P179 strain, including for example, the same S1 protein as that of the IB-QX K19-A P179 strain set forth in SEQ ID NO: 6, or a S1 protein having at least 99%, preferable at least 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%or 100%sequence identity to the S1 protein of the IB-QX K19-A P179 strain set forth in SEQ ID NO: 6, and/or the protection against virulent IB-QX challenge, including the protection against virulent IB-QX challenge in at least 80%, preferably 85%, more preferably 90%, more preferably 95%, and most preferably 100%of birds, or the improved ciliostasis score after virulent IB-QX challenge in at least 90%, preferably 95%, and most preferably 100%of birds.

9A. The attenuated IBV of clause 8A, wherein

-the descendants of the IB-QX K19-A P162 strain have the same S1 gene as that of the IB-QX K19-A P162 strain set forth in SEQ ID NO: 1, or a S1 gene having at least 99%, preferable at least 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%or 100%sequence identity to the S1 gene of the IB-QX K19-A P162 strain set forth in SEQ ID NO: 1;

-the descendants of the IB-QX K19-A P171 strain have the same S1 gene as that of the IB-QX K19-A P162 strain set forth in SEQ ID NO: 2, or a S1 gene having at least 99%, preferable at least 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%or 100%sequence identity to the S1 gene of the IB-QX K19-A P162 strain set forth in SEQ ID NO: 2; or

-the descendants of the IB-QX K19-A P179 strain have the same S1 gene as that of the IB-QX K19-A P162 strain set forth in SEQ ID NO: 3, or a S1 gene having at least 99%, preferable at least 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%or 100%sequence identity to the S1 gene of the IB-QX K19-A P162 strain set forth in SEQ ID NO: 3.

10A. The attenuated IBV of clause 8A or 9A, wherein

-the descendants of the IB-QX K19-A P162 strain comprise up to 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 passage of the IB-QX K19-A P162 strain; or the descendant of the IB-QX K19-A P162 strain is the 10^th, 9^th, 8^th, 7^th, 6^th, 5^th, 4^th, 3^rd, 2^nd or 1^st passage of the IB-QX K19-A P162 strain; or the descendant of the IB-QX K19-A P162 strain is obtainable by passaging the IB-QX K19-A P162 strain for 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 passage; preferably, the descendant of the IB-QX K19-A P162 strain is obtainable by passaging the IB-QX K19-A P162 strain for 5, 4, 3, 2 or 1 passage;

-the descendants of the IB-QX K19-A P171 strain comprise up to 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 passage of the IB-QX K19-A P171 strain; or the descendant of the IB-QX K19-A P171 strain is the 10^th, 9^th, 8^th, 7^th, 6^th, 5^th, 4^th, 3^rd, 2^nd or 1^st passage of the IB-QX K19-A P171 strain; or the descendant of the IB-QX K19-A P171 strain is obtainable by passaging the IB-QX K19-A P171 strain for 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 passage; preferably, the descendant of the IB-QX K19-A P171 strain is obtainable by passaging the IB-QX K19-A P171 strain for 5, 4, 3, 2 or 1 passage; or

-the descendants of the IB-QX K19-A P179 strain comprise up to 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 passage of the IB-QX K19-A P179 strain; or the descendant of the IB-QX K19-A P179 strain is the 10^th, 9^th, 8^th, 7^th, 6^th, 5^th, 4^th, 3^rd, 2^nd or 1^st passage of the IB-QX K19-A P179 strain; or the descendant of the IB-QX K19-A P179 strain is obtainable by passaging the IB-QX K19-A P179 strain for 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 passage; preferably, the descendant of the IB-QX K19-A P179 strain is obtainable by passaging the IB-QX K19-A P179 strain for 5, 4, 3, 2 or 1 passage.

11A. An attenuated infectious bronchitis virus (IBV) , wherein the attenuated IBV is selected from a group consisting of: the IB-QX K19-A P162 strain deposited under the deposition number CCTCC NO: V202366, any one of the descendants of the IB-QX K19-A P162 strain comprising up to 5, 4, 3, 2 or 1 passage of the IB-QX K19-A P162 strain, the IB-QX K19-A P171 strain deposited under the deposition number CCTCC NO: V202367, any one of the descendants of the IB-QX K19-A P171 strain comprising up to 5, 4, 3, 2 or 1 passage of the IB-QX K19-A P171 strain, the IB-QX K19-A P179 strain deposited under the deposition number CCTCC NO: V202368, and any one of the descendants of the IB-QX K19-A P179 strain comprising up to 5, 4, 3, 2 or 1 passage of the IB-QX K19-A P179 strain.

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

CLAUSE SET B

1B. A method of preparing an attenuated infectious bronchitis virus (IBV) , comprising a step of passaging the IB-QX K19 P7 strain deposited under the deposition number CCTCC NO: V202364 to at least the 160^th passage, preferably the 160^th to 200^th passages, more preferably, the 160^th to 180^th passages, more preferably, the 162^nd to 179^th passages, more preferably, the 162^nd, 163^rd, 164^th, 165^th, 166^th, 167^th, 168^th, 169^th, 170^th, 171^st, 172^nd, 173^rd, 174^th, 175^th, 176^th, 177^th, 178^th or 179^th passage, and most preferably, the 162^nd, 171^st or 179^th passage, on a chicken embryo or chicken embryo derived cells.

2B. The method of clause 1B, wherein the chicken embryo derived cells are primary chicken embryo cells; more preferably primary chicken embryo cells are fibroblasts or cells derived from kidney, liver or lung tissue.

3B. The method of clause 1B or 2B, wherein the attenuated IBV comprises a S1 gene comprising or consisting of a nucleotide sequence encoding an amino acid sequence having at least 95%sequence identity to the amino acid sequence set forth in SEQ ID NO: 4;

or

4B. The method of any one of clauses 1B to 3B, wherein the attenuated IBV comprises a S1 gene comprising or consisting of a nucleotide sequence having at least 95%sequence identity to the nucleotide sequence set forth in SEQ ID NO: 1, preferably having at least 96%, 97%, 98%, 99%or 100%sequence identity to the nucleotide sequence set forth 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 to the nucleotide sequence set forth in SEQ ID NO: 1, more preferably, 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 to the nucleotide sequence set forth in SEQ ID NO: 1, most preferably, having at least 99.9%or 100%sequence identity to the nucleotide sequence set forth in SEQ ID NO: 1;

or

the attenuated IBV comprises a S1 gene comprising or consisting of a nucleotide sequence selected from a group consisting of: SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3.

5B. The method of any one of clauses 1B to 4B, wherein the attenuated IBV is obtainable by passaging the IB-QX K19 P7 strain to the 162^nd passage, and the S1 gene of the attenuated IBV comprises or consists of a nucleotide sequence encoding an amino acid sequence selected from a group consisting of: SEQ ID NO: 4, preferably the S1 gene of the attenuated IBV comprises or consists of a nucleotide sequence set forth in SEQ ID NO: 1; or

wherein the attenuated IBV is obtainable by passaging the IB-QX K19 P7 strain to the 171^st passage, and the S1 gene of the attenuated IBV comprises or consists of a nucleotide sequence encoding an amino acid sequence selected from a group consisting of: SEQ ID NO: 5, preferably the S1 gene of the attenuated IBV comprises or consists of a nucleotide sequence set forth in SEQ ID NO: 2; or

wherein the attenuated IBV is obtainable by passaging the IB-QX K19 P7 strain to the 179^th passage, and the S1 gene of the attenuated IBV comprises or consists of a nucleotide sequence encoding an amino acid sequence selected from a group consisting of: SEQ ID NO: 6, the S1 gene of the attenuated IBV comprises or consists of a nucleotide sequence set forth in SEQ ID NO: 3.

6B. The method of any one of clauses 1B to 5B, wherein the attenuated IBV is selected from a group consisting of: the IB-QX K19-A P162 strain deposited under the deposition number CCTCC NO: V202366, the IB-QX K19-A P171 strain deposited under the deposition number CCTCC NO: V202367, and the IB-QX K19-A P179 strain deposited under the deposition number CCTCC NO: V202368.

7B. A method of preparing an attenuated infectious bronchitis virus (IBV) , comprising a step of:

passaging an IBV strain for within 10 passages on a chicken embryo or chicken embryo derived cells; more preferably, passaging an IBV strain for 5, 4, 3, 2 or 1 passage on a chicken embryo or chicken embryo derived cells, wherein the IBV strain is selected from a group consisting of the IB-QX K19-A P162 strain deposited under the deposition number CCTCC NO: V202366, the IB-QX K19-A P171 strain deposited under the deposition number CCTCC NO: V202367, and the IB-QX K19-A P179 strain deposited under the deposition number CCTCC NO: V202368.

8B. The method of clause 7B, wherein the chicken embryo derived cells are primary chicken embryo cells; more preferably primary chicken embryo cells are fibroblasts or cells derived from kidney, liver or lung tissue.

9B. The method of any one of clauses 1B to 8B, wherein the attenuated IBV is isolated.

CLAUSE SET C

C1. An immunogenic composition or an attenuated live vaccine against infection of an infectious bronchitis virus (IBV) , comprising a therapeutically or prophylactically effective amount of the attenuated IBV of any one of clauses in SET A, or a therapeutically or prophylactically effective amount of the attenuated IBV prepared by the method of any one of clauses in SET B;

preferably, the amount of the attenuated IBV is 1 to 10 log₁₀ EID₅₀ per ml of the attenuated IBV; more preferably, the amount of the attenuated IBV is 2 to 8 log₁₀ EID₅₀ per ml of the attenuated IBV; more preferably, the amount of the attenuated IBV is 3 to 8 log₁₀ EID₅₀ per ml of the attenuated IBV; more preferably, the amount of the attenuated IBV is 5 to 8 log₁₀ EID₅₀ per ml of the attenuated IBV; more preferably, the amount of the attenuated IBV is 6 to 7 log₁₀ EID₅₀ per ml of the attenuated IBV; and most preferably, the amount of the attenuated IBV is 6.5 log₁₀ EID₅₀ per ml of the attenuated IBV.

C2. The immunogenic composition or an attenuated live vaccine of clause C1, comprising at least a veterinary acceptable carrier;

preferably, the veterinary acceptable carrier is a lyoprotectant.

C3. The immunogenic composition or an attenuated live vaccine of any one of clauses C1 to C2, wherein the immunogenic composition or an attenuated live vaccine is formulated for a single dose administration; and/or

wherein the immunogenic composition or an attenuated live vaccine is a form of an eye drop, a nasal drop, an oculonasal drop or a spray, preferably a spray.

C4. The immunogenic composition or an attenuated live vaccine of any one of clauses C1 to C3, wherein the immunogenic composition or an attenuated live vaccine is for the prevention and/or treatment of infection of IBV in birds, preferably in poultry, more preferably in chicken.

C5. The immunogenic composition or an attenuated live vaccine of any one of clauses C1 to C4, wherein the poultry or the 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 of age; preferably, the poultry or the chicken is 1 day of age.

CLAUSE SET D

D1. A method of preparing an immunogenic composition or an attenuated live vaccine against infection of an infectious bronchitis virus (IBV) , comprising steps of:

(1) preparing the attenuated IBV of any one of clauses in SET A;

CLAUSE SET E

E1. A method of immunizing birds, or a method of preventing or preventing clinical signs caused by IBV in birds, or a method of reducing the ciliostasis in birds, wherein the method comprises administering to the subject a therapeutically or prophylactically effective amount of the attenuated IBV of any one of clauses in SET A, or a therapeutically or prophylactically effective amount of the attenuated IBV prepared by the method of any one of clauses in SET B, or a therapeutically or prophylactically effective amount of the immunogenic composition or an attenuated live vaccine of any one of clauses in SET C.

E2. Used of a therapeutically or prophylactically effective amount of the attenuated IBV of any one of clauses in SET A or a therapeutically or prophylactically effective amount of the attenuated IBV prepared by the method of any one of clauses in SET B in preparation of an immunogenic composition or an attenuated live vaccine for immunizing birds; preventing or preventing clinical signs caused by IBV in birds; or for reducing the ciliostasis in birds.

E3. A therapeutically or prophylactically effective amount of the attenuated IBV of any one of clauses in SET A, or a therapeutically or prophylactically effective amount of the attenuated IBV prepared by the method of any one of clauses in SET B, or a therapeutically or prophylactically effective amount of the immunogenic composition or an attenuated live vaccine of any one of clauses in SET C, or a therapeutically or prophylactically effective amount of the immunogenic composition or an attenuated live vaccine of prepared by the method of the clause in SET D for use in a method of immunizing birds, or for use in a method of preventing or preventing clinical signs caused by IBV in birds, or for use in a method of reducing the ciliostasis in birds, wherein the method comprises administering to the subject said therapeutically or prophylactically effective amount of said attenuated IBV, immunogenic composition or attenuated live vaccine.

E4. An immunogenic composition or an attenuated live vaccine against infection of an infectious bronchitis virus (IBV) comprising a therapeutically or prophylactically effective amount of the attenuated IBV of any one of clauses in SET A or a therapeutically or prophylactically effective amount of the attenuated IBV prepared by the method of any one of clauses in SET B, used in a method of immunizing birds, or a method of preventing or preventing clinical signs caused by IBV in birds, or a method of reducing the ciliostasis in birds.

E5. The method or or the attenuated IBV, immunogenic composition or attenuated live vaccine for use of any one of clauses E1 to E4, wherein the clinical signs caused by IBV are selected from a group consisting of: ciliostasis, rales, egg drop, kidney lesions, watery diarrhea, weight loss, virus load, viral shedding and combinations thereof;

preferably, the clinical signs caused by IBV are ciliostasis; and/or

preferably, the birds are poultry, more preferably are chicken; more preferably, the chicken is 1 day of age.

Examples

The subsequent examples further illustrate the invention in an exemplified manner. It is understood that the invention is not limited to any of those examples as described below. A person skilled in the art understands that the performance, results and findings of these examples can be adapted and applied in a broader sense in view of the general description of the invention.

Example 1 -Passaging and attenuation of the IB-QX K19 P7 strain

In 2015, a field strain (named K19 or IB-QX K19 strain) was isolated from the kidney tissues of diseased chickens in a chicken farm in Henan Province, PRC suspected of outbreaks of infectious bronchitis by Shandong Agricultural University. The descendant of this field strain after passaging for 4 passages (the 4^th passage) was purchased by BOEHRINGER INGELHEIM VETMEDICA (CHINA) CO., LTD., and then this descendant was continuously passaged to the 7^th passage on a chicken embryo. The 7^th passage of the strain was identified as an avian IBV strain with a serotype of QX, and this strain remained strong virulence. The strain that was passaged for 7 passages from the K19 strain was named the IB-QX K19 P7 strain (IB-QX represents an IBV of serotype QX, and K19 P7 represents the descendant of the K19 strain after being passaged for 7 passages) . Throughout the invention, the naming of similar virus strains has similar meanings.

The IB-QX K19 P7 strain was deposited on the date of August 4, 2003 in CHINA CENTER FOR TYPE CULTURE COLLECTION (CCTCC) , Wuhan University, Wuhan 430072, P. R. China, under accession number CCTCC NO: V202364.

The IB-QX K19 P7 strain was diluted with PBS in 1: 1000, and then was inoculated into an 8 to 10-day-old chicken embryo. After 40 hours of infection, chicken embryo was refrigerated for 24 hours at 2-8℃, subsequently allantoic fluid was collected, and then residual chicken embryo tissues were removed by centrifugation to obtain a virus strain of the 8^th passage (named the IB-QX K19 P8 strain) . The virulence test results showed that the IB-QX K19 P8 strain still remains a strong virulence. The IB-QX K19 P8 strain was used as a challenge strain in followings examples to evaluate the protective efficacy of a series of attenuated strains. The IB-QX K19 P8 strains are deposited on the date of August 4, 2003 in CCTCC, Wuhan University, Wuhan 430072, P. R. China, under accession number CCTCC NO: V202365.

The IB-QX K19 P8 strain was continuously passaged by using the above-mentioned method, and the virus strains of the 162^nd passage, the 171^st passage and the 179^th passage were prepared respectively. The safety test results in the Example 2 showed that the virulence of the strain of the 162^th passage strains had been significantly attenuated, without causing any IB-related clinical symptoms in chickens. The three virus strains were all attenuated IBV strains which were named the IB-QX K19-A P162 strain, the IB-QX K19-A P171 strain and the IB-QX K19-A P179 strain ( “A” represents “attenuated” ) respectively. Throughout the invention, the naming of similar attenuated strains has similar meanings.

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

SEQ ID NO: 1

SEQ ID NO: 4

The IB-QX K19-A P171 strain was deposited on the date of August 4, 2003 in CCTCC, Wuhan University, Wuhan 430072, P. R. China, under accession number CCTCC NO: V202367. The nucleotide sequence of the S1 gene of the IB-QX K19-A P171 strain is set forth in SEQ ID NO: 2; and the amino acid sequence encoded by the S1 gene (S1 protein) of the IB-QX K19-A P171 strain is set forth in SEQ ID NO: 5.

SEQ ID NO: 2

SEQ ID NO: 5

The IB-QX K19-A P179 strain was deposited on the date of August 4, 2003 in CCTCC, Wuhan University, Wuhan 430072, P. R. China, under accession number CCTCC NO: V202368. The nucleotide sequence of the S1 gene of the IB-QX K19-A P179 attenuated strain is set forth in SEQ ID NO: 3; and the amino acid sequence encoded by the S1 gene (S1 protein) of the IB-QX K19-A P179 strain is set forth in SEQ ID NO: 6.

SEQ ID NO: 3

SEQ ID NO: 6

The S1 genes of each two of the IB-QX K19-A P162, IB-QX K19-A P171 and IB-QX K19-A P179 strains were subjected to sequence alignment. The sequence alignment was performed using DNAMAN Software with default parameters. The results showed that the sequence identity of the S1 gene bettween the IB-QX K19-A P162 strain and the IB-QX K19-A P171 strain is 99.9%, the sequence identity of the S1 gene bettween the IB-QX K19-A P162 strain and the IB-QX K19-A P179 strain is 99.8%, and the sequence identity of the S1 gene bettween the IB-QX K19-A P171 strain and the IB-QX K19-A P179 strain is 99.9 (See Figures 1-3) . The sequence identity of the S1 protein bettween the IB-QX K19-A P162 strain and the IB-QX K19-A P171 strain is 99.8%, the sequence identity of the S1 protein bettween the IB-QX K19-A P162 strain and the IB-QX K19-A P179 strain is 99.8%, and the sequence identity of the S1 protein bettween the IB-QX K19-A P171 strain and the IB-QX K19-A P179 strain is 100.0% (See Figures 4-6) . It can be determined from the results that the nucleotide sequences of the S1 genes/proteins within a range of about 20 passgages from the 162^nd passage to the 179^th passage keep nearly no change.

Example 2 -Safety and reversion of virulence evaluations of IB-QX K19-A P162 strain

1-day-old SPF chickens were vaccinated with the IB-QX K19-A P162 strain through an oculonasal route. The tracheal tissues from SPF chickens at 5 days post vaccination were collected and prepared into a tissue homogenate. 1-day-old SPF chickens were vaccinated with the tissue homogenate, and after being continuously passaged for five times, the IB-QX K19-A P162 strain was evaluated for its safety on 1-day-old SPF chickens and its reversion of virulence after five passages.

The IB-QX K19-A P162 strain was passaged for five times in SPF chickens. Except for 10 SPF chickens used in the fifth passage, 16 SPF chickens were used in each passage during the first to the fourth passaging.

The first passaging: 1-day-old SPF chickens were inoculated with 50 μl of the IB-QX K19-A P162 strain through an oculonasal route at a dose of 10^5.5EID₅₀/chicken (single dose administration) , thus completing the first passaging (the strain after the 1^st passgaing from the IB-QX K19-A P162 strain was named P162-BP1) . Clinical observation was performed on 10 SPF chickens in this group for 10 days (Group 1) .

On 5 days post vaccination, tracheal tissues were collected from the remaining 6 SPF chickens and then prepared into a tissue homogenate as an inoculum for the next passage. The inoculum was subjected to virus isolation and identification, so as to ensure a successful passaging.

The preparation method for the tissue homogenate was as follows: about 1.5 cm of fresh tracheal tissue samples from each SPF chicken were taken and placed in a sterile centrifuge tube. The tissue samples were cut into small pieces and ground with beads, and 1 ml of PBS containing 10%antibiotics was added. The samples were shaken with a tissue homogenizer for 5 minutes at a frequency of 30 times/s; then the samples were centrifuged for 5 min at 12000 rpm/min, and then supernatant was taken. The tracheal tissue homogenate of 6 SPF chickens in each group was mixed together and used as an inoculum for the next passage.

The second, third and fourth passaging: 1-day-old SPF chickens were inoculated with 50 μl of tracheal tissue homogenate prepared from the previous passage by oculonasal route (single dose administration) , thus completing the second, third and fourth passaging (the strain after the 2^nd, the 3^rd and the 4^th passgaing was named P162-BP2, P162-BP3 and P162-BP4, respectively) . 10 SPF chickens were selected for clinical observation in each passage for 10 days (Groups 2, 3, and 4) .

Similarly, on the 5 days post vaccination, tracheal tissues were collected from the remaining 6 SPF chickens and then prepared into a tissue homogenate as an inoculum for the next passage (the preparation method of the tissue homogenate was the same as that of the first passage) .

The fifth passaging: 1-day-old SPF chickens were inoculated with 50 μl of tracheal tissue homogenate prepared from the 4^th passage by oculonasal route. 10 SPF chickens in this group were subjected to clinical observation for 21 days (Group 5) .

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, with 10 SPF chickens in each group. The SPF chickens in Group 6 were inoculated with 50 μl of the IB-QX K19-A P162 strain through an oculonasal route at a dose of 10^5.5EID₅₀/chicken. The SPF chickens in Group 7 were administered with phosphate buffer solution (PBS) through an oculonasal route. SPF chickens in each group were subjected to clinical observation for 21 days.

Table 1: Experimentation design of Example 2

“-” represents “Not available”

The results showed that after 1-day-old chickens were inoculated with the IB-QX K19-A P162 strain, and tracheal tissues prepared from each subsequent passage contained a virus amount of greater than 10^4.63 EID₅₀/mL. The IB-QX K19-A P162 strain have good adaptability to chickens and can be stably passaged in 1-day-old chickens.

Table 2: Amount of viruses in each inoculum

The results also showed that 1-day-old chickens did not show any IB-related symptoms and deadth after being inoculated with the IB-QX K19-A P162 strain. SPF chickens vaccinated with the subsequent inoculums did not show any any IB-related symptoms, and no pathological changes in any target organs were found at the autopsy, either. Both the morbidity and mortality of SPF chickens in each of five passages were 0%. The IB-QX K19-A P162 strain did not show any reversion of virulence after passaging for five passages. It can be determined from the results that the IB-QX K19-A P162 strain and its descendants within the five passages were safe for 1-day-old chickens.

Example 3 -Efficacy evaluation of the IB-QX K19-A P162 strain and the IB-QX K19 P171 strain through oculonasal route

The protective efficacy of the IB-QX K19-A P162 strain and the IB-QX K19-A P171 strain against IBV infection was evaluated.

100 1-day-old SPF chickens were randomly divided into 4 groups, with 25 chickens in each group. Groups 1 and 2 were used as immunization groups, while Group 3 was used as a challenge control group. 25 1-day-old SPF chickens from the same source and batch were used as control group (Group 4) without being vaccinated and infected.

SPF chickens in Groups 1-2 were vaccinated with 50 μl of the IB-QX K19-A P162 strain and the IB-QX K19-A P171 strain (10^4.0EID₅₀/chicken; single dose administration) through oculonasal route respectivley. SPF chickens in Group 3 were administrated with 50 μl of PBS through oculonasal route as a challenge control group.

Table 3: Experimentation design of Example 3

“-” represents “Not available”

From the day of vaccination to the day before challenge, all the SPF chickens were subjected to clinical observation once a day to record whether SPF chickens had any IB-related symptoms such as coughing, sneezing, breathing difficulties, head shaking, or tear stains.

The challenge was performed on the 21 days post vaccination. SPF chickens in Groups 1-3 were challenged with 50 μl of the IB-QX K19 P8 strain through an oculonasal route at a dose of 10^4.0EID₅₀/chicken. After challenge, the SPF chickens were continuously observed for 5 days.

After SPF chickens were subjected to challenge, all the SPF chickens were subjected to clinical observation and scoring once a day to record whether SPF chickens had any IB-related symptoms. Scoring standard: normal = 0 score, and abnormal = 1 score.

On the 5 days post challenge, all the surviving SPF chickens were euthanized and fresh tracheal tissue samples from each chicken were collected. The tracheal tissues of 5 SPF chickens in each group were selected to make tracheal rings for ciliostasis scoring.

Scoring system:

0 = if 100%of tracheal rings showed ciliary activity.

1 = if more than 75%while less than 100%of tracheal rings showed ciliary activity.

2 = if more than 50%while less than 75%of tracheal rings showed ciliary activity.

3 = if more than 25%while less than 50%of tracheal rings showed ciliary activity.

4 = if less than 25%of tracheal rings showed ciliary activity or showed no ciliary activity.

The chicken will be determined as being infected with IBV (IBV positive) if the score of two or more of tracheal rings is > 2.

During the experimentation, clinical observation was conducted once a day, and Groups 1-5 were continuously observed for 27 days and recorded.

After vaccination, all the SPF chickens in each groups showed no clinical abnormalities, with good health.

After challenge, no clinical abnormalities were observed in SPF chickens in Groups 1-2, and the SPF chickens had no gross pathological changes in autopsy. On the 5 days post challenge, the tracheal tissue samples were collected and were subjected to IBV isolation. The positive rates of tracheal samples of 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.

Table 4: Protection rate in each group

Note 1: “-” represents “Not available” .

Note 2: “a” shows clinical observation results after challenge; “b” shows gross pathological observation results of autopsy; and “c” shows the positive rate of the virus.

On the 5 days post challenge, SPF chickens in Groups 1-3 were subjected to tracheal ring cilia scoring. The results showed that the number of SPF chickens with tracheal ring score of > 2 was 0/5, 0/5 and 5/5, respectively. The incidence rates of SPF chickens in each group were 0%, 0%, and 100%, respectively.

It can be determined from the above results that the IB-QX K19-A P162 strain and the IB-QX K19-A P171 strain can provide superior protective efficacy againt IBV infection in SPF chickens.

Example 4 -Efficacy evaluation of the IB-QX K19-A P162 strain, the IB-QX K19-A P171 strain and the IB-QX K19 P179 strain through a spray route

The protective efficacy of the IB-QX K19-A P162 strain, the IB-QX K19-A P171 strain and the IB-QX K19 P179 strain against IBV infection was evaluated.

120 1-day-old SPF chickens were randomly divided into 4 groups, with 30 chickens in each group. Groups 1-3 were used as immunization groups, and Group 4 was used as a challenge control group.

SPF chickens in Groups 1-3 were vaccinated with the IB-QX K19-A P162 strain, the IB-QX K19-A P171 strain and the IB-QX K19 P179 strain (10^5.0EID₅₀/chicken; single dose administration) through a spray route respectivley. SPF chickens in Group 4 were administrated with PBS through a spray route as a chanlledge control group.

Table 5: Experimentation design of Example 4

Note 1: “-” represents “Not available” .

The challenge was performed on the 21 days post vaccination. SPF chickens in Groups 1-4 were challenged with the IB-QX K19 P8 strain through a nasal dropping route at a dose of 10^4.0EID₅₀/chicken (50 μl) .

On the 5 days post challenge, 20 SPF chickens were selected from each group and were euthanized. Fresh tracheal tissue samples from each chicken were collected for virus isolation. On the 6 days post challenge, the rest 10 SPF chickens in each group were euthanized, and their tracheal tissues were collected to make tracheal rings for ciliostasis scoring. The standard of the ciliostasis scoring is the same as the Example 3.

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

Table 6: Protection rates of the three attanuated strains

The results also showed that the number of SPF chickens in Groups 1-4 with tracheal ring score of > 2 was 0/10, 2/10, 0/10 and 10/10, respectively. The incidence rates of SPF chickens in each group were 0%, 20%, 0%, and 100%.

Table 7: Incidence rates of SPF chickens

The above results demonstrate that all of the IB-QX K19-A P162 strain, the IB-QX K19-A P171 strain and the IB-QX K19-A P179 strain could provide sufficient protection efficacy for 1-day-old chickens, and all the three attenuated strains could be used for the development of a vaccine against IBV infection. Moreover, the efficacy of the three attenuated strains also meets the requirements of large-scale spray immunization in chicken farms. Compared with the oculonasal route, spray route does not require a lot of manpower and greatly shortens the vaccination time.

The above results also demonstrate that, within the range of at least about 20 passages starting from the IB-QX K19-A P162 strain, the IB-QX K19-A P171 strain and the IB-QX K19 P179 strain could still achieve superior immune protection efficacy against the IBV infection. That is, a broad range of descendants could be derived from the IB-QX K19-A P162 strain and the IB-QX K19-A P171 strain, and these descendants are all effective to provide immune protections against the IBV infection. Without bound to any theory, the wide range of descendants may be resulted from the high sequence identity among the S1 gene/protein sequences from the IB-QX K19-A P162 strain to the IB-QX K19-A P179 strain. The wide range of descendants can ensure a wider passage window period during the production, thereby being convenient for large-scale industrial production.

Example 5 -Efficacy evaluation of the IB-QX K19-A P162 strain under a lower vaccination dose

The protective efficacy of the IB-QX K19-A P162 strain against IBV infection under a lower vaccination dose was evaluated.

60 1-day-old SPF chickens were randomly divided into 5 groups, with 12 chickens in each group. Groups 1-3 were used as immunization groups, Group 4 was used as a challenge control group, and Group 5 was used as a negative control group. SPF chickens in Groups 1-3 were vaccinated with the IB-QX K19-A P162 strain with doses of 10^5.0EID₅₀/chicken, 10^4.0EID₅₀/chicken and 10^3.0EID₅₀/chicken (single dose administration) through a spray route respectivley. SPF chickens in Groups 4-5 were administrated with PBS through a spray route.

The challenge was performed on the 21 days post vaccination. SPF chickens in Groups 1-4 were challenged with the IB-QX K19 P8 strain through a nasal dropping route at a dose of 10^4.0EID₅₀/chicken (50 μl) . On the 5 days post challenge, all SPF chickens from each group were euthanized. Fresh tracheal tissue samples from each chicken were collected to make tracheal rings for ciliostasis scoring. The standard of the ciliostasis scoring is the same as the Example 3.

Table 8: Experimentation design of Example 5

“-” represents “Not available”

The above results showed that the IB-QX K19-A P162 strain could still provide sufficient protection efficacy for 1-day-old chickens through a spray route when its dose is lowered to 10^3.0EID₅₀/chicken. It further demonstrates that the IB-QX K19-A P162 strain has superior immunogenicity and can provide full protection againt IBV infection in chickens.

SEQUENCE LISTINGS:

SEQ ID NO: 1 -the nucleotide sequence of the S1 gene of the IB-QX K19-A P162 strain

SEQ ID NO: 2 -the nucleotide sequence of the S1 gene of the IB-QX K19-A P171 strain

SEQ ID NO: 3 -the nucleotide sequence of the S1 gene of the IB-QX K19-A P179 strain

SEQ ID NO: 4 -the amino acid sequence of the S1 protein of the IB-QX K19-A P162 strain

SEQ ID NO: 5 -the amino acid sequence of the S1 protein of the IB-QX K19-A P171 strain

SEQ ID NO: 6 -the amino acid sequence of the S1 protein of the IB-QX K19-A P179 strain

Claims

An attenuated infectious bronchitis virus (IBV) , wherein the attenuated IBV comprises a S1 gene comprising or consisting of a nucleotide sequence encoding an amino acid sequence having at least 95%sequence identity to the amino acid sequence set forth in SEQ ID NO: 4;

preferably, wherein the attenuated IBV comprises a S1 gene comprising or consisting of a nucleotide sequence encoding an amino acid sequence having at least 96%, 97%, 98%, 99%or 100%sequence identity to the amino acid sequence set forth in SEQ ID NO: 4;

more preferably, wherein the attenuated IBV comprises a S1 gene comprising or consisting 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 to the amino acid sequence set forth in SEQ ID NO: 4;

or

the attenuated IBV comprises a S1 gene comprising or consisting of a nucleotide sequence encoding an amino acid sequence selected from a group consisting of: SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6.
The attenuated IBV of claim 1, wherein the S1 gene comprises or consists of a nucleotide sequence having at least 95%sequence identity to the nucleotide sequence set forth in SEQ ID NO: 1;

preferably, wherein the S1 gene comprises or consists of a nucleotide sequence having at least 96%, 97%, 98%, 99%or 100%sequence identity to the nucleotide sequence set forth in SEQ ID NO: 1;

more preferably, wherein the S1 gene comprises or consists 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 to the nucleotide sequence set forth in SEQ ID NO: 1;

or

the S1 gene comprises or consists of a nucleotide sequence selected from a group consisting of: SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3.
The attenuated IBV of claim 1 or 2, wherein the attenuated IBV is of a QX serotype; preferably wherein the attenuated IBV is derived from the IB-QX K19 P7 strain deposited under the deposition number CCTCC NO: V202364.
The attenuated IBV of any one of claims 1 to 3, wherein the attenuated IBV is at least the 160^th passage; preferably, the 160^th to the 200^th passages; more preferably, the 160^th to the 180^th passages; more preferably, the 162^nd to 179^th passages; more preferably, the 162^nd passage, the 163^rd passage, the 164^th passage, the 165^th passage, the 166^th passage, the 167^th passage, the 168^th passage, the 169^th passage, the 170^th passage, the 171^st passage, the 172^nd passage, the 173^rd passage, the 174^th passage, the 175^th passage, the 176^th passage, the 177^th passage, the 178^th passage or the 179^th passage; and most preferably, the 162^nd passage, the 171^st passage or the 179^th passage.
An attenuated infectious bronchitis virus (IBV) , wherein the attenuated IBV is obtainable by passaging the IB-QX K19 P7 strain deposited under the deposition number CCTCC NO: V202364 to at least the 160^th passage; preferably the 160^th to 200^th passages; more preferably, the 160^th to 180^th passages; more preferably, the 162^nd to 179^th passages; more preferably, the 162^nd, 163^rd, 164^th, 165^th, 166^th, 167^th, 168^th, 169^th, 170^th, 171^st, 172^nd, 173^rd, 174^th, 175^th, 176^th, 177^th, 178^th or 179^th passage; and most preferably, the 162^nd, 171^st or 179^th passage.
The attenuated IBV of claim 5, wherein the attenuated IBV comprises a S1 gene comprising or consisting of a nucleotide sequence encoding an amino acid sequence having at least 95%sequence identity to the amino acid sequence set forth in SEQ ID NO: 4;

preferably, wherein the attenuated IBV comprises a S1 gene comprising or consisting of a nucleotide sequence encoding an amino acid sequence having at least 96%, 97%, 98%, 99%or 100%sequence identity to the amino acid sequence set forth in SEQ ID NO: 4;

more preferably, wherein the attenuated IBV comprises a S1 gene comprising or consisting 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 to the amino acid sequence set forth in SEQ ID NO: 4;

or

the attenuated IBV comprises a S1 gene comprising or consisting of a nucleotide sequence encoding an amino acid sequence selected from a group consisting of: SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6.
The attenuated IBV of claim 5 or 6, wherein the S1 gene comprises or consists of a nucleotide sequence having at least 95%sequence identity to the nucleotide sequence set forth in SEQ ID NO: 1;

preferably, wherein the S1 gene comprises or consists of a nucleotide sequence having at least 96%, 97%, 98%, 99%or 100%sequence identity to the nucleotide sequence set forth in SEQ ID NO: 1;

more preferably, wherein the S1 gene comprises or consists 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 to the nucleotide sequence set forth in SEQ ID NO: 1;or

the S1 gene comprises or consists of a nucleotide sequence selected from a group consisting of: SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3.
An attenuated infectious bronchitis virus (IBV) ,

wherein the attenuated IBV is the IB-QX K19-A P162 strain deposited under the deposition number CCTCC NO: V202366 and its descendants, and the descendants of the IB-QX K19-A P162 strain are any attenuated descendant IBVs thereof having all of the identifying characteristics of the deposited IB-QX K19-A P162 strain, including for example, the same S1 protein as that of the IB-QX K19-A P162 strain set forth in SEQ ID NO: 4, or a S1 protein having at least 99%, preferable at least 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%or 100%sequence identity to the S1 protein of the IB-QX K19-A P162 strain set forth in SEQ ID NO: 4, and/or the protection against virulent IB-QX challenge, including the protection against virulent IB-QX challenge in at least 80%, preferably 85%, more preferably 90%, more preferably 95%, and most preferably 100%of birds, or the improved ciliostasis score after virulent IB-QX challenge in at least 90%, preferably 95%, and most preferably 100%of birds;

wherein the attenuated IBV is the IB-QX K19-A P171 strain deposited under the deposition number CCTCC NO: V202367 and its descendants, and the descendants of the IB-QX K19-A P171 strain are any attenuated descendant IBVs thereof having all of the identifying characteristics of the deposited IB-QX K19-A P171 strain, including for example, the same S1 protein as that of the IB-QX K19-A P171 strain set forth in SEQ ID NO: 5, or a S1 protein having at least 99%, preferable at least 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%or 100%sequence identity to the S1 protein of the IB-QX K19-A P171 strain set forth in SEQ ID NO: 5, and/or the protection against virulent IB-QX challenge, including the protection against virulent IB-QX challenge in at least 80%, preferably 85%, more preferably 90%, more preferably 95%, and most preferably 100%of birds, or the improved ciliostasis score after virulent IB-QX challenge in at least 90%, preferably 95%, and most preferably 100%of birds; or

wherein the attenuated IBV is the IB-QX K19-A P179 strain deposited under the deposition number CCTCC NO: V202368 and its descendants, and the descendants of the IB-QX K19-A P179 strain are any attenuated descendant IBVs thereof having all of the identifying characteristics of the deposited IB-QX K19-A P179 strain, including for example, the same S1 protein as that of the IB-QX K19-A P179 strain set forth in SEQ ID NO: 6, or a S1 protein having at least 99%, preferable at least 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%or 100%sequence identity to the S1 protein of the IB-QX K19-A P179 strain set forth in SEQ ID NO: 6, and/or the protection against virulent IB-QX challenge, including the protection against virulent IB-QX challenge in at least 80%, preferably 85%, more preferably 90%, more preferably 95%, and most preferably 100%of birds, or the improved ciliostasis score after virulent IB-QX challenge in at least 90%, preferably 95%, and most preferably 100%of birds.
The attenuated IBV of claim 8, wherein

- the descendants of the IB-QX K19-A P162 strain have the same S1 gene as that of the IB-QX K19-A P162 strain set forth in SEQ ID NO: 1, or a S1 gene having at least 99%, preferable at least 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%or 100%sequence identity to the S1 gene of the IB-QX K19-A P162 strain set forth in SEQ ID NO: 1;

- the descendants of the IB-QX K19-A P171 strain have the same S1 gene as that of the IB-QX K19-A P162 strain set forth in SEQ ID NO: 2, or a S1 gene having at least 99%, preferable at least 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%or 100%sequence identity to the S1 gene of the IB-QX K19-A P162 strain set forth in SEQ ID NO: 2; or

- the descendants of the IB-QX K19-A P179 strain have the same S1 gene as that of the IB-QX K19-A P162 strain set forth in SEQ ID NO: 3, or a S1 gene having at least 99%, preferable at least 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%or 100%sequence identity to the S1 gene of the IB-QX K19-A P162 strain set forth in SEQ ID NO: 3.
The attenuated IBV of claim 8 or 9, wherein

- the descendants of the IB-QX K19-A P162 strain comprise up to 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 passage of the IB-QX K19-A P162 strain; or the descendant of the IB-QX K19-A P162 strain is the 10^th, 9^th, 8^th, 7^th, 6^th, 5^th, 4^th, 3^rd, 2^nd or 1^st passage of the IB-QX K19-A P162 strain; or the descendant of the IB-QX K19-A P162 strain is obtainable by passaging the IB-QX K19-A P162 strain for 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 passage; preferably, the descendant of the IB-QX K19-A P162 strain is obtainable by passaging the IB-QX K19-A P162 strain for 5, 4, 3, 2 or 1 passage;

- the descendants of the IB-QX K19-A P171 strain comprise up to 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 passage of the IB-QX K19-A P171 strain; or the descendant of the IB-QX K19-A P171 strain is the 10^th, 9^th, 8^th, 7^th, 6^th, 5^th, 4^th, 3^rd, 2^nd or 1^st passage of the IB-QX K19-A P171 strain; or the descendant of the IB-QX K19-A P171 strain is obtainable by passaging the IB-QX K19-A P171 strain for 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 passage; preferably, the descendant of the IB-QX K19-A P171 strain is obtainable by passaging the IB-QX K19-A P171 strain for 5, 4, 3, 2 or 1 passage; or

- the descendants of the IB-QX K19-A P179 strain comprise up to 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 passage of the IB-QX K19-A P179 strain; or the descendant of the IB-QX K19-A P179 strain is the 10^th, 9^th, 8^th, 7^th, 6^th, 5^th, 4^th, 3^rd, 2^nd or 1^st passage of the IB-QX K19-A P179 strain; or the descendant of the IB-QX K19-A P179 strain is obtainable by passaging the IB-QX K19-A P179 strain for 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 passage; preferably, the descendant of the IB-QX K19-A P179 strain is obtainable by passaging the IB-QX K19-A P179 strain for 5, 4, 3, 2 or 1 passage.
An attenuated infectious bronchitis virus (IBV) , wherein the attenuated IBV is selected from a group consisting of: the IB-QX K19-A P162 strain deposited under the deposition number CCTCC NO: V202366, any one of the descendants of the IB-QX K19-A P162 strain comprising up to 5, 4, 3, 2 or 1 passage of the IB-QX K19-A P162 strain, the IB-QX K19-A P171 strain deposited under the deposition number CCTCC NO: V202367, any one of the descendants of the IB-QX K19-A P171 strain comprising up to 5, 4, 3, 2 or 1 passage of the IB-QX K19-A P171 strain, the IB-QX K19-A P179 strain deposited under the deposition number CCTCC NO: V202368, and any one of the descendants of the IB-QX K19-A P179 strain comprising up to 5, 4, 3, 2 or 1 passage of the IB-QX K19-A P179 strain.
The attenuated IBV of any one of claims 1-11, wherein the attenuated IBV is isolated.
A method of preparing an attenuated infectious bronchitis virus (IBV) , comprising a step of:

passaging an IBV strain for within 10 passages on a chicken embryo or chicken embryo derived cells; more preferably, passaging an IBV strain for 5, 4, 3, 2 or 1 passage on a chicken embryo or chicken embryo derived cells, wherein the IBV strain is selected from a group consisting of the IB-QX K19-A P162 strain deposited under the deposition number CCTCC NO: V202366, the IB-QX K19-A P171 strain deposited under the deposition number CCTCC NO: V202367, and the IB-QX K19-A P179 strain deposited under the deposition number CCTCC NO: V202368.
The method of claim 13, wherein the chicken embryo derived cells are primary chicken embryo cells; more preferably primary chicken embryo cells are fibroblasts or cells derived from kidney, liver or lung tissue.
An immunogenic composition or an attenuated live vaccine against infection of an infectious bronchitis virus (IBV) , comprising a therapeutically or prophylactically effective amount of the attenuated IBV of any one of claims 1 to 12, or a therapeutically or prophylactically effective amount of the attenuated IBV prepared by the method of any one of claims 13 to 14;

preferably, the amount of the attenuated IBV is 1 to 10 log₁₀ EID₅₀ per ml of the attenuated IBV; more preferably, the amount of the attenuated IBV is 2 to 8 log₁₀ EID₅₀ per ml of the attenuated IBV; more preferably, the amount of the attenuated IBV is 3 to 8 log₁₀ EID₅₀ per ml of the attenuated IBV; more preferably, the amount of the attenuated IBV is 5 to 8 log₁₀ EID₅₀ per ml of the attenuated IBV; more preferably, the amount of the attenuated IBV is 6 to 7 log₁₀ EID₅₀ per ml of the attenuated IBV; and most preferably, the amount of the attenuated IBV is 6.5 log₁₀ EID₅₀ per ml of the attenuated IBV.
The immunogenic composition or an attenuated live vaccine of claim 15, comprising at least a veterinary acceptable carrier;

preferably, the veterinary acceptable carrier is a lyoprotectant.
The immunogenic composition or an attenuated live vaccine of any one of claims 15 to 16, wherein the immunogenic composition or an attenuated live vaccine is formulated for a single dose administration; and/or

wherein the immunogenic composition or an attenuated live vaccine is a form of an eye drop, a nasal drop, an oculonasal drop or a spray, preferably a spray.
The immunogenic composition or an attenuated live vaccine of any one of claims 15 to 17, wherein the immunogenic composition or an attenuated live vaccine is for the prevention and/or treatment of infection of IBV in birds, preferably in poultry, more preferably in chicken.
The immunogenic composition or an attenuated live vaccine of any one of claims 15 to 18, wherein the poultry or the 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 of age; preferably, the poultry or the chicken is 1 day of age.
A method of immunizing birds, or a method of preventing or preventing clinical signs caused by IBV in birds, or a method of reducing the ciliostasis in birds, wherein the method comprises administering to the subject a therapeutically or prophylactically effective amount of the attenuated IBV of any one of claims 1 to 12, or a therapeutically or prophylactically effective amount of the attenuated IBV prepared by the method of any one of claims 13 to 14, or a therapeutically or prophylactically effective amount of the immunogenic composition or an attenuated live vaccine of any one of claims 15 to 19.
A therapeutically or prophylactically effective amount of the attenuated IBV of any one of claims 1 to 12, or a therapeutically or prophylactically effective amount of the attenuated IBV prepared by the method of any one of claims 13 to 14, or a therapeutically or prophylactically effective amount of the immunogenic composition or an attenuated live vaccine of any one of claims 15 to 19 for use in a method of immunizing birds, or for use in a method of preventing or preventing clinical signs caused by IBV in birds, or for use in a method of reducing the ciliostasis in birds, wherein the method comprises administering to the subject said therapeutically or prophylactically effective amount of said attenuated IBV, immunogenic composition or attenuated live vaccine.
The method or the attenuated IBV, immunogenic composition or attenuated live vaccine for use of claim 21, wherein the clinical signs caused by IBV are selected from a group consisting of: ciliostasis, rales, egg drop, kidney lesions, watery diarrhea, weight loss, virus load, viral shedding and combinations thereof;

preferably, the clinical signs caused by IBV are ciliostasis; and/or

preferably, the birds are poultry, more preferably are chicken; more preferably, the chicken is 1 day of age.