A gi-13 lineage avian infectious bronchitis virus attenuated strain and application thereof

Abstract

The application discloses a GI-13 lineage chicken infectious bronchitis virus attenuated strain and application thereof, and belongs to the technical field of attenuated vaccines. In order to provide a safe and effective GI-13 lineage IBV attenuated vaccine strain, the application is named as LGX-B strain. The application has good safety for chickens of any age, and can stimulate effective immune protection after immunization of the chickens. The application provides a GI-13 lineage chicken infectious bronchitis virus attenuated strain, which is preserved in the China General Microbiological Culture Collection Center on November 26, 2025, and the preservation number is CGMCC NO. 47087. The application is used for in-vitro proliferation of candidate viruses required by chicken infectious bronchitis vaccines, diagnostic preparations and the like, and has important practical application value.

Description

Technical Field

[0001] This invention belongs to the field of attenuated vaccine technology, specifically relating to a GI-13 lineage of attenuated chicken infectious bronchitis virus and its application. Background Technology

[0002] Infectious bronchitis (IB) is a major acute respiratory infectious disease of chickens caused by the infectious bronchitis virus (IBV), and is one of the most significant infectious diseases facing the poultry industry worldwide. Since its discovery in the 1930s, IBV has caused substantial economic losses to the poultry industry annually, making it a major problem for poultry farmers globally. This disease can cause morbidity and mortality in chickens of different ages and breeds, posing an extremely serious threat. Its main manifestations are fourfold: 1) Infection in chicks causes severe morbidity and mortality, with some strains exhibiting mortality rates exceeding 80%, resulting in direct economic losses; 2) Viral infection creates conditions for secondary infections by bacteria, mycoplasma, and other pathogens, easily leading to mixed infections and thus higher morbidity and mortality rates, increasing the culling rate; 3) Viral infection in broilers causes slowed weight gain and reduced feed utilization; 4) Viral infection in laying hens causes permanent damage to the reproductive system, resulting in "water sac chickens" and "false hens," and infection in adult hens leads to a decline in egg production and quality. Therefore, IBV poses a significant threat to the poultry industry and is a crucial disease problem that urgently needs to be addressed in my country and worldwide.

[0003] Currently, there are at least 11 lineages (genotypes) of IBV circulating in my country. The main circulating lineages include: GI Type 19(QX) and GI Type 22(HN08), GI Type 7(TW), GI Type 13(4 / 91), GI Type 13 (4 / 91), GI-28, GI-29, GVI 1(TC07 2) Type and GVII-1, etc., different lineages have certain differences in genomic characteristics, pathogenicity and epidemiological characteristics.

[0004] Vaccination is the most effective means of preventing and controlling this disease. Currently, commercially available IBV vaccines include live attenuated vaccines and inactivated vaccines. Live vaccines are the primary vaccine for preventing infectious bronchitis in chickens. However, because IBV is a highly mutable RNA virus, its main antigenic genes frequently change during genetic evolution, leading to the continuous emergence of new viruses. The antigenicity of these new viruses often drifts, resulting in a lack of cross-immunity or poor cross-protection between different virus types. This is a major reason why, despite the continued use of the traditional GI-1 lineage (Mass type) live vaccine since the 1980s, mutated circulating strains continue to infect chicken flocks. In recent years, my country has successively developed GI-19 and GI-22 lineage live vaccines targeting the antigenic characteristics of circulating strains in my country, and has also introduced GI-13 lineage live vaccines adapted to circulating strains from abroad through various means. However, given the complex and diverse infectious bronchitis strains currently circulating in my country, these commercially available vaccines cannot fully cover the antigenic protection spectrum of all strains. The cross-protection coverage of each vaccine against non-homologous strains is limited and lacks significant regularity. Therefore, each live vaccine is primarily used for the prevention of antigen-matched homologous wild-type strains. Among these, the GI-13 lineage strain has been prevalent in Europe, including the UK and France, since the 1990s, gradually becoming the dominant circulating strain. In response, Europe developed a live attenuated vaccine (strain 4 / 91) against the prevalent wild-type GI-13 lineage strain to control its spread. It is noteworthy that subsequent evaluation studies found that the 4 / 91 live attenuated vaccine has strong virulence, persisting in the chicken's body with a shedding period of over 40 days. While continuously stimulating the immune system to produce immunity, this prolonged infection in target organs and continuous attenuated virus replication further damage the integrity of the upper respiratory tract, making it susceptible to secondary bacterial infections. On the other hand, the continued presence of the virus in the body provides more opportunities for gene recombination between the virus and other types of strains or vaccine strains. This is also an important reason why the GI-13 lineage strains prevalent in China are mainly recombinant viruses produced by recombination of the S gene of the GI-13 lineage strain with the genome or GI-1 lineage genome framework prevalent in China. The first instance of this type of strain being found circulating in my country was in 2003. As my country's poultry industry introduced the European GI-13 lineage (4 / 91 type) vaccine in various ways, recombinant strains of the GI-13 lineage with the circulating strains in my country gradually became widespread in different regions of my country. Even immunization with the 4 / 91 type vaccine could not completely protect against the infection and spread of these recombinant viruses in chicken flocks. The pathogenicity of these circulating strains differs from that of other viruses currently predominantly causing nephropathic effects, such as GI-19, GI-22, and GI-28. Although there is no obvious mortality after infection, respiratory damage is severe, especially in young chickens, which are more susceptible to secondary mixed infections with other pathogens, amplifying the harm and losses caused by the disease.To date, various research institutions in my country have developed domestic attenuated vaccines against this prevalent strain, including the currently publicly available NNA strain and FNO-E55 strain, which are attenuated GI-13 lineage chicken infectious bronchitis virus vaccine strains developed in my country. However, despite their commercial application, these vaccine strains are basically consistent with the European 4 / 91 strain in terms of infection characteristics and immune protection. That is, they have persistent infection in the respiratory and digestive tracts for a long time, with a viral shedding period of 36 to 40 days, relatively strong virulence, greater damage to the respiratory ciliated epithelium, significantly reduced ciliary activity after infection, and a long duration of infection.

[0005] As is well known, the best attenuated live vaccines for infectious bronchitis in chickens are characterized by good immunogenicity while maintaining excellent safety. Classic examples include the GI-1 lineage attenuated live vaccines (such as H120 and Ma5), which can replicate and multiply in the chicken respiratory tract for a short period, eliciting an immune response and being cleared from the body within 12-16 days without further viral shedding. However, currently available GI-13 lineage attenuated live vaccines do not possess this characteristic of short-term replication in the chicken respiratory tract and often cause severe damage to the respiratory ciliated epithelium. This significantly limits the safe application of GI-13 lineage-type infectious bronchitis vaccines. Therefore, there is an urgent need to cultivate safer attenuated strains of the GI-13 lineage that do not cause persistent damage to the respiratory tract of chickens to adapt to the immunization against this type of prevalent strain in my country. Especially given that the current prevalent GI-13 lineage in China is mainly composed of recombinant viruses formed by recombination of GI-13 and GI-19 lineage strains, screening and cultivating targeted, safe and effective attenuated vaccine strains is an important problem that urgently needs to be solved in the field of IBV research. Summary of the Invention

[0006] The purpose of this invention is to provide a GI-13 lineage IBV attenuated vaccine strain adapted to chicken embryos, exhibiting excellent safety in chickens. Compared to previous attenuated strains of the same type, the strain developed in this invention significantly shortens the viral shedding period after immunization of chickens and significantly improves the protection rate against the GI-13 lineage virus prevalent in my country.

[0007] This invention provides a GI-13 lineage of attenuated strain of infectious bronchitis virus (IBV), named attenuated strain LGX-B, which was deposited at the China General Microbiological Culture Collection Center on November 26, 2025, with accession number CGMCC NO. 47087.

[0008] This invention provides a microbial preparation of the above-mentioned attenuated strain of GI-13 lineage chicken infectious bronchitis virus and its derivative viruses.

[0009] The present invention provides an attenuated vaccine composition comprising the above-mentioned attenuated strain of GI-13 lineage chicken infectious bronchitis virus or the above-mentioned microbial preparation.

[0010] Further specifying, the attenuated vaccine composition is a monovalent vaccine, a bivalent vaccine, or a multivalent vaccine.

[0011] Further specifying, the attenuated vaccine composition also includes an oily adjuvant or an immune complex adjuvant.

[0012] Further specified, the infectious bronchitis virus content in chickens is ≥10. 6.0 EID 50 / 0.1ml.

[0013] This invention provides the application of the above-mentioned attenuated vaccine composition in the preparation of a drug for the prevention of infectious bronchitis in chickens.

[0014] This invention provides the application of the above-mentioned GI-13 lineage attenuated strain of infectious bronchitis virus in chickens or the above-mentioned microbial preparation in the preparation of a vaccine for the prevention of infectious bronchitis in chickens.

[0015] Furthermore, the vaccine can produce immune protection after immunizing chicken embryos or chickens.

[0016] Beneficial Effects: This invention uses the typical recombinant representative strain CK / CH / LGX / 231104 of the GI-13 lineage, a dominant strain of infectious bronchitis virus (IBV) prevalent in China in recent years, as the parent virus. It differs from previously commercially used classic GI-1, GI-19, and GI-22 lineage strains, and also differs significantly in genomic structure from European-origin GI-13 lineage strains. This is especially true for strains belonging to the GI-13 lineage, which exhibit low replication levels in chicken embryos and are relatively difficult to passage. This invention utilizes chicken embryo cold adaptation passage technology to screen for attenuated GI-13 lineage IBV strains capable of effectively infecting and massively replicating in chicken embryos. These strains exhibit stable passage in chicken embryos and higher potency. The chicken embryo-adapted infectious bronchitis virus strain obtained in this invention can be used as a seed virus for preparing a live attenuated vaccine to prevent the GI-13 lineage of infectious bronchitis variant recombinant virus prevalent in China. Compared with similar live attenuated strains, the viral shedding period after immunization is significantly shortened, highlighting its excellent safety. The protection rate against the recombinant virus prevalent in my country is significantly improved, highlighting its excellent efficacy. It can be used as a candidate strain for in vitro propagation of viruses required for chicken infectious bronchitis vaccines, diagnostic reagents, etc., and has significant practical application value.

[0017] [Bio-deposit Information]: A GI-13 lineage of infectious bronchitis virus (IBV), specifically the attenuated IBV strain LGX-B, was deposited on November 26, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO. 47087. The deposit address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. Detailed Implementation

[0018] The parental wild-type strain CK / CH / LGX / 231104 is represented by GenBank entry KT852992.1.

[0019] The H120 strain is a commercially available GI-1 lineage vaccine strain that is currently widely used in the market.

[0020] The LDT3-A strain is a commercially available vaccine strain of the GI-22 lineage that was previously cultured and screened by the inventors.

[0021] Strain M41 is the recognized model virus for infectious bronchitis virus (IBV) and the first typical strain discovered since its discovery. It is widely referred to as the Mass type virus (GI-1 lineage). For details of our research on this virus, please see Lingfeng Chen, Tingting Zhang, Zongxi Han, Xiangang Kong, Shengwang Liu. Molecular and antigenic characteristics of Massachusetts genotype infectious bronchitis coronavirus in China. Veterinary Microbiology, 2015;181(3-4):241-251.

[0022] Strain 4 / 91 is a live attenuated vaccine strain of the GI-13 lineage IBV prevalent in Europe, which differs significantly from the strains prevalent in my country, with marked differences in genotype and serotype. For details of our research, please see: Tingting Zhang, Zongxi Han, Qianqian Xu, Qiuling Wang, Mengying Gao, Wei Wu, Yuhao Shao, Huixin Li, Xiangang Kong, Shengwang Liu. Serotype shift of a 793 / B genotype infectious bronchitis coronavirus by natural recombination. Infection, Genetics and Evolution. 2015, 32:377-87.

[0023] NNA strain and FNO-E55 strain are GI-13 lineage infectious bronchitis virus strains bred by other units in my country. Their S1 gene and genome structure are highly similar to those of the 4 / 91 vaccine strain (over 99%). Related research reports can be found in: (1) Li S, Chen W, Shen Y, Xia J, Fan S, Li N, Luo Y, Han X, Cui M, Zhao Y, Huang Y. Molecular characterization of infectious bronchitis virus in Southwestern China for the protective efficacy evaluation of four live vaccine strains. Vaccine. 2022 Jan 21;40(2):255-265. (2) Huang Songjian, Zhang Yun, Huang Mengjiao, et al. Evaluation of the immunoprotective efficacy of the attenuated live vaccine NNA for infectious bronchitis in yellow chickens. Poultry Farming and Disease Control, 2018,(08):25-30. LGX / 100508, I0718 / 17, I0722 / 17, I0724 / 17, and I0737 / 17 are epidemic strains of infectious bronchitis in chickens from the GI-13 lineage isolated by our team. Their S1 gene and genomic structure are highly similar (over 99%) to 4 / 91 vaccine strains. Related research reports can be found in the literature described here: Liwen Xu, Mengting Ren, Jie Sheng, Tianxin Ma, Zongxi Han, Yan Zhao, Junfeng Sun, Shengwang Liu. Genetic and biological characteristics of four novel recombinant avianinfectious bronchitis viruses isolated in China. Virus Research 263(2019)87-97 ck / CH / LBJ / 140413, ck / CH / LHB / 130927, ck / CH / LLN / 130102, ck / CH / LSD / 110851, ck / CH / LBJ / 140402, ck / CH / LLN / 130101, HLJ / 111246, ck / CH / LSD / 110410, ck / CH / LHB / 121010, ck / CH / LHB / 130630, ck / CH / LHB / 120141, ck / CH / LHB / 140532, and ck / CH / LSD / 110851 are epidemic strains of GI-13 lineage chicken infectious bronchitis isolated by our team. Their S1 gene and genome structure are highly similar (over 99%) to 4 / 91 vaccine strains. Related research reports can be found in: Zongxi Han, Wenjun Zhao. Yuqiu Chen, Qianqian Xu, Junfeng Sun, Tingting Zhang, Yan Zhao, Shuling Liang, Mengying Gao, Qiuling Wang, Xiangang Kong, Shengwang Liu. Genetic, antigenic, and pathogenic characteristics of avian infectious bronchitis viruses genetically related to 793 / B in China. Veterinary Microbiology 203 (2017) 125–135. Example 1. Obtaining GI-13 lineage chicken infectious bronchitis virus adapted to replication and proliferation in SPF chicken embryos. Development of chicken embryo-adapted strain (LGX-B): 1) The CK / CH / LGX / 231104 strain was obtained in 2023 from a diseased chicken flock collected from a chicken farm in Guangxi. After grinding, freezing and thawing, and filtering to sterilize the sample in the laboratory, it was inoculated into 9-day-old SPF chicken embryos, and the allantoic fluid was harvested. RT-PCR and nucleic acid sequencing analysis confirmed the isolation of a GI-13 lineage infectious bronchitis virus. The allantoic fluid was purified using limiting dilution, and exogenous virus testing confirmed the purity of the isolated virus, which was named the CK / CH / LGX / 231104 strain. This virus is a representative GI-13 lineage strain prevalent in chicken flocks in my country. Infection of 1-day-old SPF chickens can cause 100% morbidity and stimulate the production of high levels of antibodies. The strain exhibits good immunogenicity and is a highly virulent parent strain for preparing attenuated vaccine strains.

[0024] 2) Cold adaptation subculture of IBV strain CK / CH / LGX / 231104 in SPF chicken embryos: The highly virulent IBV strain CK / CH / LGX / 231104 was subjected to continuous gradient cooling cold adaptation subculture using 9-day-old SPF chicken embryos. During subculture, two chicken embryos were inoculated into the allantoic cavity, with 0.1 ml inoculated into each embryo. The subculture conditions were as follows: 10 generations (generations 1-10) were started at 37℃, with each generation incubated for 36-36℃. After 72 hours, the chicken embryos were cooled at 2–8°C for 18–24 hours. The embryos were observed for disease development, and the allantoic fluid was aseptically collected for the next passage. Starting from the 11th generation, the incubation temperature was lowered to 35°C and passaged for 10 consecutive generations (11–20 generations). Then, the incubation temperature was maintained at 31°C for another 10 generations (21–30 generations), and finally, the incubation temperature was maintained at 29°C for another 10 generations (31–40 generations). During this period, every 10 generations, the seed virus was returned to 37°C for one generation and retained as the seed virus. After passage to the 40th generation, the embryos were continuously passaged for 8 generations (41–48 generations) under incubation conditions at 37°C to obtain the attenuated vaccine strain LGX-B of this invention (48th generation). During the passage of the virus, as the number of passages increases, the virus becomes more and more adaptable to chicken embryos, and the lesions in the chicken embryos gradually become more obvious. Chicken embryos inoculated with the virus show obvious developmental disorders (dwarf embryos), curled into a ball shape, embryonic hemorrhage, amniotic edema and thickening and other typical IBV infection lesions.

[0025] 3) Purity test of the passaged virus: Seed viruses from passages P1, P10, P20, P30, P40, and P48 were used to determine the agglutination characteristics of the virus solution on 1% SPF chicken peripheral blood erythrocytes. The results showed that none of the viruses from these six passages agglutinated chicken peripheral blood erythrocytes, indicating that the chicken embryo allantoic fluid did not contain other viruses that could cause agglutination of chicken peripheral blood erythrocytes. Simultaneously, the seed viruses from passages P40 and P48 were subjected to sterility testing, mycoplasma testing, and exogenous virus testing according to the appendix of the current Chinese Veterinary Pharmacopoeia, Part III. The results showed that both P40 and P48 (LGX-B strain) were free from bacterial, fungal, and mycoplasma contamination, as well as exogenous virus contamination, indicating that the virus was pure. The 48th generation was named LGX-B and preserved.

[0026] Example 2. Potency, pathogenicity, and efficacy of LGX-B strain 1) Titer determination of different passaged viruses: Seed viruses from passages P1, P10, P20, P30, P40, and P48 were serially diluted 10-fold. Four suitable dilutions were selected and inoculated into five 9-10 day old SPF chicken embryos, 100 µL per embryo. The embryos were incubated at 37°C for 7 days, and the infection and survival numbers of the embryos were recorded daily. The EID was calculated using the Reed-Muench method. 50 The results showed that the titer of the P1 generation of the parental circulating virus strain CK / CH / LGX / 231104 was 10. 4.0 EID 50 / 0.1ml, P10 generation titer 10 4.7 EID 50 / 0.1ml, P20 generation titer 10 5.3 EID 50 / 0.1ml, P30 generation titer 10 6.3 EID 50 / 0.1ml, P40 and P48 generations are 10 6.5 EID 50 / 0.1ml. As the virus is passaged in chicken embryos, it gradually adapts to the embryos, and the viral titer gradually increases after passage, stabilizing in the P40 and P48 generations and reaching its highest viral titer.

[0027] 2) Pathogenicity evaluation of LGX-B strain: 75 1-2 day old SPF chickens were randomly divided into 5 groups of 10 each, and were isolated and housed with free access to feed and water. Groups 1-4 were treated with P1 generation (10... 4.0 EID 50 P20 (10) 5.3 EID 50 P40 (10) 4.8 EID 50 ) and P48 (106.5 EID 50 Group 1 received 100µL of PBS via intranasal inoculation; Group 5 served as the control group, receiving 100µL of PBS via intranasal inoculation. The morbidity of the vaccinated chickens was observed and recorded daily after inoculation. Five days post-inoculation, five chickens from each group were euthanized, and trachea were collected from both vaccinated and control chickens to create tracheal rings. The ciliary activity and cessation of movement were observed in each segment of the tracheal ring under an inverted microscope. Results showed that the pathogenicity of the CK / CH / LGX / 231104 strain to 1-day-old SPF chicks gradually decreased with increasing passage number after continuous passage in chicken embryos. Chickens in generations P3 through P40 showed varying degrees of morbidity and mortality, with a morbidity rate of 1 / 10 to 10 / 10. The main symptoms of diseased chickens included head shaking, neck retraction, and open-mouth breathing. Only one chicken in generation P40 exhibited short-term, occasional head shaking, while no clinical symptoms or mortality were observed in generation P48. Autopsy revealed no tissue lesions, consistent with the SPF control group.

[0028] Table 1. Pathogenicity of chicken embryo virus strain CK / CH / LGX / 231104 at different passages to 1-day-old SPF chickens.

[0029] 3) Evaluation of the immunogenicity of LGX-B strain: Sixty 1-day-old SPF chickens were randomly divided into 4 groups of 15 chickens each. Two groups of chicks were nasally vaccinated with LGX-B, and 10 groups were vaccinated with LGX-B. 3.0 EID 50 / bird; the other two groups of chicks served as the control group, and were injected nasally or ocularly with normal chicken embryo allantoic fluid, 0.1ml / bird. The disease incidence of the vaccinated flocks was observed and recorded daily, and serum was collected from each group on days 14 and 20 post-immunization. Antibody seroconversion was determined using an IBV antibody detection kit. Simultaneously, on days 14 and 20 post-immunization, one group of vaccinated chickens and one group of control chickens were selected and injected 10 / bird nasally. 6.0 EID 500.1 ml of strain I0722 / 17 virus was administered to each chicken, and the disease incidence was observed and recorded for 14 days. Five days after challenge, oropharyngeal swabs were collected from immunized and control chickens for virus isolation and RT-PCR detection. Simultaneously, tracheal rings were collected from immunized and control chickens in each group, and tracheal rings were constructed. The ciliary activity and cessation of movement in each segment of the tracheal ring were observed under an inverted microscope. The degree of ciliary cessation was scored as follows: 4 points for more than 75% ciliary cessation; 3 points for 50%–75% (inclusive); 2 points for 25%–50% (inclusive); 1 point for 25% or less; and 0 points for no ciliary cessation. Results showed that the antibody seroconversion rates at 14 and 20 days post-immunization were 5 / 10 and 10 / 10, respectively. The control group chickens remained antibody-negative throughout the entire treatment period. Fourteen days post-immunization, all immunized chickens achieved clinical protection of 10 / 10, but the oropharyngeal shedding rate was 5 / 10, and only 60% (3 / 5) of the chickens had a tracheal ciliary activity score below 20 (protected), failing to meet the industry-recognized 80% protection standard. Twenty days post-immunization, the immunized chickens achieved complete clinical protection with an oropharyngeal shedding rate of 2 / 10 and a tracheal ciliary activity score below 5 (protected), resulting in an overall protection rate of 80%.

[0030] Table 2. Immunopotency data of LGX-B strain at different time points after immunization.

[0031] The P48 generation of strain CK / CH / LGX / 231104 (LGX-B strain) was selected as a candidate strain for vaccine development. The LGX-B strain was submitted for preservation, with the following accession number: CGMCC No. 47087; classification: Infectious Bronchitis Virus; preservation date: November 26, 2025; depositary institution: China General Microbiological Culture Collection Center (CGMCC); depositary address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences.

[0032] Example 3. Replication kinetics of LGX-B strain 1. Infection and sampling of LGX-B strain: The LGX-B strain virus solution was diluted 1000 times and inoculated into 9- to 10-day-old SPF chicken embryos. The embryos were incubated at 37°C and the embryo fluid was harvested at 12, 24, 36, 48, 60 and 72 hours after incubation.

[0033] 2. Virus titer determination: Virus fluid (chicken embryo fluid) harvested at different times was serially diluted 10-fold with sterile physiological saline. A suitable dilution was selected, and 0.1 mL of the diluted solution was inoculated into 5 SPF chicken embryos per embryo. The embryos were incubated at 37°C, and candled daily. After 7 days of incubation, embryo lesions were observed, and the EID of the virus was calculated using the Reed-Muench method. 50 The results showed that as the virus proliferated in SPF chicken embryos, the stronger the virus's ability to multiply, the higher the virus titer, reaching its peak at 60–72 hours (Table 3).

[0034] Table 3. Virus content of LGX-B strain at different time points after inoculation into chicken embryos.

[0035] 3. Stability of CK / CH / LGX / 231104 strain and characteristics of its passaged derivative viruses 1) Passaging of CK / CH / LGX / 231104 strain: The CK / CH / LGX / 231104 strain virus was continuously passaged in SPF chicken embryos up to generation P48. Each generation of virus infecting SPF chicken embryos caused significant embryo lesions, indicating that the virus, after adapting to the chicken embryo, maintains a stable ability to infect chicken embryos. Viruses from different generations were collected and stored at –70℃ for later use.

[0036] 2) Virulence evaluation of progeny viruses derived from the CK / CH / LGX / 231104 strain: Thirty one-day-old SPF chickens were randomly divided into three groups (10 chickens per group). The chickens were housed in three separate negative pressure isolators with free access to feed and water. Groups 1 and 2 were treated with the 30th generation (10-fold) progeny virus of the CK / CH / LGX / 231104 strain. 5.8 EID 50 ) and the 40th generation (10 6.0 EID 50 The chicks were inoculated with 100µL of the virus via intranasal drops per bird; the third group of chicks was not inoculated and served as a blank control group. The morbidity of the inoculated flocks was observed and recorded daily after inoculation. Results showed that no obvious disease was observed in 1-day-old chickens infected with the progeny virus of the CK / CH / LGX / 231104 strain. At the end of the observation period, no tracheal hemorrhage or specific pathological changes in the kidneys were observed during necropsy, consistent with the blank control SPF chickens.

[0037] 3) Changes in the genome of CK / CH / LGX / 231104 strain after different generations: The complete genome of the virus, representing the 30th and 40th generations of progeny strains derived from CK / CH / LGX / 231104 strain, was amplified by RT-PCR. Then, the genes were cloned and sequenced, and compared with the CK / CH / LGX / 231104 strain before subculture. The results showed that the differences between the derived progeny strains and the parent virulent strain included varying degrees of nucleotide mutations in gene segments 1ab, S, 3a, 3b, 3c, M, and N. The rate of change of these genes remained within 1%, and these gene changes did not affect the characteristics of virus infection in SPF chicken embryos.

[0038] 4) Characteristics of CK / CH / LGX / 231104 strain: CK / CH / LGX / 231104 strain is a coronavirus, and its replication characteristics exhibit typical coronavirus features. It has its own unique characteristics. During viral replication and proliferation, it relies on the virus's own RNA polymerase. This polymerase lacks proofreading function. Therefore, gene mutations during viral passage and reproduction are a common phenomenon in this type of virus. The mutation rate of the genome of the progeny viruses derived from the CK / CH / LGX / 231104 strain after passage in this invention is maintained within 1%, and the replication characteristics and attenuated virulence of the virus are preserved.

[0039] Example 4. Serotype cross-reactivity between LGX-B strain and existing GI-13 and GI-1 lineage vaccines To evaluate the antigenic cross-correlation between LGX-B strain and attenuated / vaccine strains of the GI-13 lineage, as well as commonly used GI-1 lineage vaccine strains in China, cross-neutralization tests were performed using single-factor sera from each strain. The neutralizing titers of heterologous and homologous sera were measured, and antigenic correlation was compared based on the cross-neutralization titers of different representative strains. The antigenic correlation coefficient was calculated using immunological methods: (R) = (1 / 2) × 100%, where r1 = heterologous serum titer¹ / homologous serum titer¹, r2 = heterologous serum titer² / homologous serum titer². R > 80% indicates the same type; R between 25% and 80% indicates different subtypes of the same type; and R < 25% indicates different types. The results showed that the correlation coefficients of the LGX-B strain with the attenuated vaccine strains (4 / 91, FNO-E55 and NNA) belonging to the same GI-13 lineage were all greater than 75%, indicating that they belong to the same serotype. However, the correlation coefficients with the classic vaccine strain H120 of the GI-1 lineage were all less than 25%, indicating that they belong to different serotypes.

[0040] Table 4. Results of cross-neutralization tests of LGX-B strain with other strains of the GI-13 lineage and vaccine strains of the GI-1 lineage.

[0041] Table 5. Cross-correlation coefficients (R values) between LGX-B strain and other strains of the GI-13 lineage and vaccine antigens of the GI-1 lineage.

[0042] Example 5. Comparison of the replication and shedding cycle of LGX-B strain with existing GI-13 and GI-1 lineage vaccines in chickens. 1. Duration of viral shedding and positivity rate are indicators for evaluating the safety of attenuated live vaccines for respiratory diseases. Short-term viral shedding and rapid clearance by the body effectively prevent persistent respiratory damage and reduce the risk of viral spread, which are key characteristics of high-quality attenuated live vaccines. Regarding oropharyngeal shedding, the LGX-B strain exhibits typical short-term replication and rapid clearance characteristics. The positivity rate is 7 / 10 at 4 and 8 days post-immunization, indicating effective short-term colonization in the respiratory tract, sufficient to stimulate a specific immune response. The positivity rate rapidly decreases to 4 / 10 after 12 days, and only 2 / 10 remains at 16 days. Shedding completely ceases at 20 days and subsequently at 36 and 40 days, indicating complete clearance by the chicken's respiratory system, perfectly meeting the safety standard of 12-16 days for the classic H120 vaccine to clear viral shedding. In contrast, other GI-13 strains exhibit longer shedding periods and lower clearance efficiency. For example, the 4 / 91 and FNO-E55 strains still show oropharyngeal shedding positivity rates of 1 / 10 and 2 / 10 respectively after 20 days, while the NNA strain still shows oropharyngeal shedding in 3 / 10 chickens after 20 days, significantly increasing the risk of persistent respiratory damage and vaccine virus dissemination. Regarding cloacal shedding, the LGX-B strain's peak shedding rate is concentrated at 8 days post-immunization (10 / 10), rapidly declining to 6 / 10 at 12 days, only 2 / 10 at 20 days, and completely ceasing shedding by 36 days, showing a short overall shedding period and rapid decline. In contrast, the 4 / 91, FNO-E55, and NNA strains have longer shedding durations, maintaining a 100% cloacal shedding positivity rate from 12 to 16 days, still reaching 8 / 10 at 20 days, and continuing to show 1 / 10 to 3 / 10 shedding individuals even after 36 to 40 days. Based on the comprehensive detoxification data, it is evident that LGX-B is the only strain among the tested GI-13 lineage strains that achieves short-term, efficient replication and rapid clearance from the body, overcoming the inherent safety shortcoming of the long detoxification cycle of this lineage strain.

[0043] Table 6. Comparison of viral shedding time between LGX-B strain, other strains of the GI-13 lineage, and GI-1 lineage vaccine.

[0044] 2. Chicken embryo passage and proliferation capacity is a core foundation for large-scale vaccine production. Virus strains with high titers and strong passage stability can effectively reduce vaccine production costs and ensure stable vaccine potency. Existing GI-13 lineage strains generally suffer from weak chicken embryo proliferation capacity and difficulty in passage to high titers, significantly limiting the industrial application of this type of vaccine. Based on chicken embryo passage and proliferation data for GI-13 lineage strains, the LGX-B strain only requires 48 passages to achieve 10... 7.5 EID 50 The viral titer of / ml was the highest among all tested GI-13 lineage strains. In comparison, strain 4 / 91, after more than 100 passages, FNO-E55 after 56 passages, and NNA after more than 50 passages, had viral titers of only 10. 6.5 10 6.2 10 6.0 EID 50 / ml, although it has been passaged more times, its proliferation titer is lower and its proliferation efficiency is not as good as LGX-B. Meanwhile, LGX-B's chicken embryo proliferation titer is also higher than that of the more embryo-adapted strain I0722 / 17 (10 7.0 The LGX-B strain (EID50 / ml) exhibits excellent adaptability and proliferation potential in chicken embryos. This advantage means that the LGX-B strain can quickly obtain high-titer virus stock solution without long-term, high-generation domestication, which greatly improves production efficiency and solves the pain points of weak proliferation ability and high-titer passage difficulty of traditional GI-13 lineage strains.

[0045] Table 7. Data on chicken embryo propagation levels of GI-13 lineage strains.

[0046] Example 6. Optimal selection of GI-13 lineage strains based on chicken embryo adaptability and respiratory tract detoxification characteristics. 1) Optimization based on chicken embryo adaptability indicators: Addressing the common issues of prolonged viral shedding cycles in chickens with GI-13 strains of infectious bronchitis (IBC), leading to persistent respiratory damage, cross-infection, and poor safety profiles, as well as the weak embryo adaptability and low viral titers of this strain's weak-read vaccines, this study systematically screened and adapted representative GI-13 strains (CK / CH / LGX / 231104, etc.) in the strain library to chicken embryos. Viral titers before and after embryo passage were measured. Simultaneously, the embryo-adapted viral stock solution was inoculated into 1-day-old SPF chickens, and the viral positivity rate of oropharyngeal and cloacal swabs was detected at different time points after inoculation. Results showed that the viral titer of each GI-13 strain before embryo passage was 10. 3.0 EID 50 / 0.1ml~10 4.5 EID 50 / 0.1ml, after 40-55 passages, the viral titer significantly increased to 10. 5.0 EID 50 / 0.1ml~10 6.5 EID 50 / 0.1ml. The LGX-B strain alone can reach 10 after only 48 generations of chicken embryo passage adaptation. 6.5 EID 50 The high viral titer of 0.1 ml represents the highest titer level among all tested strains, demonstrating its advantage in production suitability.

[0047] 2) Optimization based on post-vaccination viral shedding indicators in chickens: A comparison of viral shedding data from the oropharynx and cloaca at different time points in 1-day-old chickens after passage of GI-13 lineage strains showed that the LGX-B strain exhibited significantly better in vivo clearance than other GI-13 lineage strains, consistent with the viral shedding patterns of classic safe attenuated vaccines. Specifically, in oropharyngeal shedding, the LGX-B strain exhibited typical short-term replication and rapid clearance characteristics. The viral positivity rate was 7 / 10 at 4 and 8 days post-immunization, effectively achieving short-term colonization in the respiratory tract and stimulating a specific immune response. After 12 days of immunization, the viral positivity rate rapidly decreased to 4 / 10, remaining at only 2 / 10 at 16 days, and completely ceasing shedding by 20 days. In contrast, other GI-13 lineage strains had longer shedding periods and lower clearance efficiency, with shedding periods of 20 and 24 days respectively. Regarding cloacal shedding, LGX-B exhibits a cloacal shedding cycle of only 20 days, while other strains have a longer shedding period of 32-40 days. Based on both respiratory and cloacal shedding data, LGX-B is clearly an ideal candidate for a high-quality attenuated strain among the tested GI-13 lineage strains, possessing short-term respiratory shedding, rapid in vivo clearance, strong embryo compatibility, and high proliferation titer.

[0048] Table 8. Proliferation of different GI-13 strains in chicken embryos and time of viral shedding in the respiratory tract of inoculated chickens.

[0049] Example 7. Cross-immune protection of LGX-B strain against virulent strains of different lineages 1) Clinical observation: 20 days after immunizing 5-day-old SPF chickens with LGX-B strain, chickens were challenged with representative virulent strains from different lineages. The results showed that most immunized chickens challenged with heterologous strains obtained clinical protection. In contrast, chickens in the control group all had varying degrees of disease or death. Diseased chickens showed symptoms such as ruffled feathers, retracted neck, closed eyes, loss of appetite or reduced appetite, serous or catarrhal nasal discharge, and head shaking / scratching of the nose. Among them, no more than one chicken in tl / CH / LDT3 / 03 (GI-22 lineage), CK / CH / LDL / 091022 (GI-19 lineage), and CK / CH / LDL / 140520 (GI-7 lineage) had clinical symptoms, while some control group chickens died.

[0050] 2) Oropharyngeal Shedding and Tracheal Injury: In the LGX-B strain immunization group, the shedding protection rate of the prevalent GI-13 strain I0722 / 17 reached 80%, demonstrating good protective efficacy. However, the shedding protection rate of other strains after challenge was 0-30%, significantly lower than the 80% protection standard, indicating poor cross-protection with other strains. Regarding tracheal ciliary activity scores, all control group chickens had tracheal activity scores above 20. In chickens challenged with I0722 / 17, 100% had tracheal activity scores below 5. However, after challenge with M41 and tl / CH / LDT3 / 03, 20% and 40% of chickens, respectively, had scores below 20, while other immunized chickens all had scores above 20. This indicates that LGX-B strain immunization is most effective against strains within the same lineage, exhibiting significant differences in immune characteristics compared to other viral lineages, suggesting that LGX-B strain possesses a unique immunoprotective advantage within the GI-13 lineage. (A total score of >20 indicates that tracheal ciliary activity is affected, while a score of ≤20 indicates that tracheal ciliary activity is not affected; in the same industry, 80% protection is considered to be effective protection against infectious bronchitis in chickens.) Table 9. Immunization protection against different types of virulent strains using LGX-B strain.

[0051] Example 8. Evaluation of the immunogenicity of LGX-B strain in chicks 1) Evaluation test of humoral immunity efficacy of immune activation: Thirty 5-day-old SPF chickens were randomly divided into two groups (15 chickens in each group) and housed in negative pressure isolators. The chicks had free access to feed and water. The chicks in the first group were treated with LGX-B strain (10 3.0 EID 50 The chicks in the first group were vaccinated, with 100µL administered via eye drops and nasal drops per chick; the second group of chicks was not vaccinated and served as the control group. Twenty days after immunization, serum samples were collected from each group and antibody tests were performed according to the instructions of the chicken infectious bronchitis virus ELISA antibody detection kit. The results showed that all chickens were positive for antibodies, while all chickens in the control group were negative.

[0052] 2) Clinical protection evaluation: 20 days after immunization, the two groups of chicks were treated with the virulent strain I0722 / 17 (10 6.0 EID 50 The chickens were challenged by intranasal administration of 0.1 ml of the virus, 100 µL per chicken; after challenge, the incidence of disease in the flock was observed and recorded daily. The results showed that no clinical symptoms were observed in the immunized group, while 2 / 10 of the control group developed the disease.

[0053] 3) Evaluation of oropharyngeal viral shedding protection: On the 5th day after challenge in step 2), pharyngeal swabs were collected from two groups of chickens. Sterile saline containing antibiotics was added, and after shaking and filtration sterilization, 9-day-old SPF chicken embryos were inoculated. After 7 days of culture, the viral shedding of pharyngeal swabs was determined based on the presence of dwarf embryos, short embryos, and other specific chicken embryo lesions of infectious bronchitis virus. The results showed that the viral shedding of pharyngeal swabs in the LGX-B strain immunized group was 1 / 10, while that in the control group was 10 / 10. It can be seen that immunization with LGX-B strain elicits a good immune response and has a good immunoprotective effect against the circulating strains of the GI-13 lineage.

[0054] 4) Evaluation of tracheal ciliary activity: Five days after challenge, trachea were collected from five immunized chickens and five control chickens. Tracheal rings were constructed, and the ciliary activity and cessation of movement in each segment of the tracheal ring were observed under an inverted microscope. The results showed that the ciliary activity scores of the tracheal rings in the immunized group were all below 5, indicating that the tracheal cilia of the immunized chickens were well protected. In contrast, the ciliary activity scores of the tracheal rings in the control group were all above 20, indicating that the chickens were not protected.

[0055] Table 10. Results of Immunopotency Evaluation of LGX-B Strain

[0056] Example 9. Application of LGX-B strain as a seed virus for preparing different vaccines I. Preparation of LGX-B strain monovalent, bivalent, and multivalent vaccines Vaccine stock solution preparation: LGX-B strain was diluted 10-fold with sterile physiological saline and inoculated into well-developed SPF chicken embryos. The embryos were incubated at 37°C. The virus-containing embryo fluid was harvested 48–72 hours after incubation and stored at 2–8°C. Sterility testing was performed simultaneously, and the virus content was determined to be ≥10-. 6.0 EID 50 / 0.1ml.

[0057] Preparation of monovalent and bivalent live vaccines: (1) When preparing monovalent live vaccines, the virus culture medium that has passed the sterility test and virus content determination is mixed with sucrose gelatin protectant at a ratio of 8.5:1 (virus liquid: protectant). The freeze-drying protectant should be done at 40℃~50℃ (8% gelatin, 40% sucrose protectant, sterilized at 115℃ for 40 minutes). During the addition process, the virus liquid should be shaken continuously and mixed thoroughly to obtain the vaccine stock solution. The vaccine stock solution is aseptically dispensed into quantitative portions, quickly freeze-dried under vacuum, and sealed to obtain the monovalent live vaccine, which is then stored below -15℃. (2) When preparing bivalent live vaccines, the virus culture medium of the Newcastle disease vaccine attenuated La Sota strain that has passed the sterility test and virus content determination is mixed with the LGX-B strain proliferated virus liquid and then prepared according to the above embryo vaccine scheme.

[0058] Preparation of inactivated vaccines: (1) When preparing monovalent inactivated vaccines, after mixing the virus culture medium that has passed the sterility test and virus content determination, formaldehyde solution is added for inactivation to make the final concentration 0.2%, and then shaken thoroughly. Then it is placed in a shaker at 37℃ for inactivation. After inactivation is completed overnight, the inactivated virus solution is stored at 2-8℃ and should not exceed 2 months. Mix 94 parts of white oil for injection and 6 parts of Span-80, add 2% aluminum stearate, and autoclave to prepare the oil phase. Add 6% of the inactivated virus solution to Tween-80 and shake thoroughly to completely dissolve the Tween to prepare the aqueous phase. Take 3 parts of the oil phase and place it in a colloid mill. Start the motor to stir, and then slowly add 1 part of the aqueous phase. Start timing. The emulsification interval is 10, and emulsification is carried out at 4000r / m for 8 minutes. Before the emulsification is terminated, add 1% thimerosal preservative with a final concentration of 0.01%. The emulsified qualified vaccine is dispensed into sterile vaccine bottles, sealed, and the inactivated vaccine is prepared and stored at 2-8℃. (2) When preparing the bivalent inactivated vaccine, the virus solution that has passed the sterility test, virus content test, and inactivation test after the Newcastle disease vaccine strain has been proliferated is mixed with the virus solution of the LGX-B strain proliferated and then prepared according to the above vaccine preparation plan. (3) When preparing the trivalent inactivated vaccine, the virus culture solution that has passed the sterility test, virus content test, and inactivation test after the Newcastle disease vaccine strain and the egg drop syndrome vaccine strain have been proliferated and then mixed with the virus solution of the LGX-B strain proliferated and then prepared according to the above vaccine preparation plan.

[0059] II. Safety verification of vaccines prepared using the LGX-B strain: 1. Experimental Vaccines: A monovalent live LGX-B strain vaccine, batch number 202501, was prepared according to the live vaccine preparation method described in Example 2 above. 1000 doses / vial. A bivalent live LGX-B strain vaccine and the Newcastle disease vaccine strain La Sota were prepared, batch number 202502. 1000 doses / vial. Vaccine storage conditions and shelf life: Store below -15℃, shelf life is 24 months. Simultaneously, a monovalent inactivated LGX-B strain vaccine, batch number 202503, was prepared according to the inactivated vaccine preparation method described in Example 2. A bivalent inactivated LGX-B strain vaccine and the Newcastle disease vaccine strain La Sota were prepared, batch number 202504. A trivalent inactivated LGX-B strain vaccine, combined with the Newcastle disease vaccine strain La Sota and the egg drop syndrome vaccine strain, was prepared, batch number 202505. 250 doses / vial. Vaccine storage conditions and shelf life: Store at 2~8℃, shelf life is 12 months.

[0060] 2. Safety verification of live vaccine: Groups 1 and 2 (10 chicks / group) of 5-day-old SPF chicks were inoculated with batches 202501 and 202502 of live vaccine, respectively, with 10 doses (approximately 0.03-0.05 ml) administered intranasally to each chick. Group 3 of 5-day-old SPF chicks was not vaccinated and served as a control. The clinical manifestations of the chicks in each group were continuously observed after immunization, and the incidence of disease was recorded.

[0061] 3. Verification of the safety and virulence reversion of live vaccine: Fifteen 5-day-old SPF chickens were inoculated via intranasal drip with batch 202501 vaccine (LGX-B strain), with each chicken receiving a dose of 5 × 10⁻⁶. 5.5 EID 50 Five chickens were kept in a negative pressure isolator for observation. Five chickens were euthanized five days after inoculation, and their tracheas were aseptically extracted, ground, and homogenized with PBS. After three freeze-thaw cycles, the homogenate was centrifuged at 6000 rpm for 5 minutes, and the supernatant was filtered through a 0.22 μm filter membrane for sterilization. The virus content of the filtrate was determined using 9-10 day old SPF chicken embryos. Subculture was then performed on the next generation of chickens, with an inoculation dose of 0.5 ml / chick. The remaining 10 chickens were observed for 14 days after inoculation for clinical symptoms and mortality. This process was repeated for four more generations, with 10 control chickens of the same age in each generation. The S1 protein gene sequence was determined by RT-PCR in the last generation of subcultures to verify the virulence reversion safety of the LGX-B strain.

[0062] 4. Safety Verification of Inactivated Vaccine: Groups 1-3 (5 chicks / group) of 1-2 month old SPF chicks were vaccinated with batches 202503, 202504, and 202505 of vaccine, respectively, with each chick receiving two doses (approximately 1.0 ml) via intramuscular injection in the pectoral region. Group 4 of 1-2 month old SPF chicks received 1.0 ml of sterile saline via intramuscular injection in the pectoral region. Clinical manifestations of the chicks in each group were continuously observed post-immunization, and morbidity was recorded. After 14 days of observation, the injection sites were examined to observe vaccine absorption.

[0063] 5. The experimental results are shown in Table 11: (1) Safety test of live vaccine: 20 days after immunization, the two batches of live vaccine immunization groups showed no clinical symptoms of infectious bronchitis such as head shaking, lethargy, and ruffled feathers, consistent with the blank control group. During the observation period, 0 / 10 cases of disease occurred, and all groups showed normal performance. This indicates that the monovalent live vaccine prepared by LGX-B strain and the bivalent live vaccine prepared in combination with Newcastle disease vaccine strain are both safe after immunization of chickens.

[0064] Table 11 Safety test data of monovalent and bivalent live vaccines against infectious bronchitis in chickens.

[0065] (2) Results of live vaccine virulence reversion and safety verification: No clinical symptoms of infectious bronchitis virus infection, such as depression, neck retraction, arched back, rough feathers, drooping wings, and open-mouth breathing, were observed in any of the test chickens inoculated with each generation of the virus within 14 days of observation. The fifth-generation virus from batch 202501 of the vaccine was identical to the S1 protein gene sequence of the LGX-B strain. The above results indicate that the vaccine strain prepared from the LGX-B strain will not exhibit reversion of virulence during propagation and passage in chickens, and its genetic characteristics are stable, making it suitable for the preparation of live vaccines for the prevention and control of infectious bronchitis virus in the GI-13 lineage chickens.

[0066] Table 12 Clinical observation results of LGX-B strain chicken passages 1-5

[0067] (3) Safety test of inactivated vaccine: Chickens immunized with batches 202503, 202504, and 202505 were observed for 14 days. Consistent with the blank control group, all groups showed normal performance during the observation period, and no adverse reactions occurred. Autopsy revealed no obvious inflammatory reactions such as granulomas at the injection site, indicating good vaccine absorption. This demonstrates that the monovalent inactivated vaccine prepared from the LGX-B strain, the bivalent vaccine prepared with the Newcastle disease vaccine strain, and the trivalent inactivated vaccine prepared with the Newcastle disease vaccine and egg drop syndrome vaccine are all safe after immunization.

[0068] III. Verification of the Immunogenic Efficacy of Vaccine Prepared from LGX-B Strain 1. Experimental Vaccines: A monovalent live vaccine of the LGX-B strain was prepared according to the live vaccine preparation method of Example 4, batch number 202501, 1000 doses / vial. A bivalent live vaccine of the LGX-B strain and the Newcastle disease vaccine strain La Sota was prepared, batch number 202502, 1000 doses / vial. Vaccine storage conditions and shelf life: Store below -15℃, shelf life is 24 months. Simultaneously, a monovalent inactivated vaccine of the LGX-B strain was prepared according to the inactivated vaccine preparation method of Example 2, batch number 202503. A bivalent inactivated vaccine of the LGX-B strain and the Newcastle disease vaccine strain La Sota was prepared, batch number 202504. A trivalent inactivated vaccine of the LGX-B strain and the Newcastle disease vaccine strain La Sota and the egg drop syndrome vaccine strain was prepared, batch number 202405, 500 doses / vial. Vaccine storage conditions and shelf life: Store at 2~8℃, shelf life is 12 months.

[0069] 2. Usage and Dosage: For live vaccines, dilute with physiological saline according to the dosage indicated on the label. Use a dropper to administer one drop (approximately 0.03–0.05 ml) to each chicken's nostril. For inactivated vaccines, administer 0.3–0.5 ml intramuscularly per chicken using a syringe.

[0070] 3. Verification of live vaccine efficacy: Five-day-old SPF chicks in groups 1 and 2 (15 chicks / group) were inoculated with live vaccine batches 202501 and 202502, respectively, with one dose (approximately 0.03-0.05 ml) administered intranasally to each chick. Ten five-day-old SPF chicks in group 3 served as a control group, without vaccination. Twenty days post-immunization, all chicks were challenged intranasally with the virulent strain I0722 / 17 (containing 10... 6.0 EID 50 Five days after challenge, 10 pharyngeal swabs were collected from each group for virus isolation. Tracheal rings were prepared from 5 chickens, and tracheal ciliary activity was scored. Clinical morbidity was continuously observed in each group. Simultaneously, 30-day-old SPF chicks in group 4 (10 chicks / group) were vaccinated with batch 202502 live vaccine, with each chick receiving 0.01 dose (approximately 0.03–0.05 ml) via intranasal drip. Group 5, 30-day-old SPF chicks, served as a control and were not vaccinated. Fourteen days post-immunization, chickens in groups 4 and 5 were challenged with the Beijing strain of Newcastle disease (10-10 ... 4.0 ELD 50 The strain was highly virulent, and the incidence and mortality of chickens in each group were continuously observed (Table 10) for 14 days.

[0071] 4. Verification of inactivated vaccine efficacy: One-month-old SPF chicks in groups 1-3 (5 chicks / group) were vaccinated with batches 202503, 202504, and 202505 of vaccine, respectively, with each chick receiving one dose (approximately 0.5 ml) via intramuscular injection in the pectoral region. One-month-old SPF chicks in group 4 received 0.5 ml of sterile saline solution via intramuscular injection in the pectoral region. Twenty-eight days post-immunization, all four groups of chicks were challenged with I0722 / 17 (10...) via intramuscular injection. 5.5 EID 50 Five days after challenge, chickens in each group were culled, and trachea samples were collected from each chicken. The supernatant from the homogenized sample was inoculated into 9-day-old SPF chicken embryos for virus isolation. Groups 5 and 6 (5 chickens / group) of 30-day-old SPF chicks were inoculated with batches 202504 and 202505 of inactivated vaccine, with each chick receiving one dose (approximately 0.5 ml). Group 7 of 30-day-old SPF chickens received 0.5 ml of sterile saline solution per chick. Fourteen days post-immunization, groups 5 to 7 were challenged with the Newcastle disease Beijing strain (10) via intramuscular injection. 4.0 ELD 50 The strain was highly virulent, and the morbidity and mortality of chickens in each group were continuously observed for 14 days. Group 8 (5 chickens / group) of 30-day-old SPF chicks were vaccinated with batch 202505 of inactivated vaccine, with each chick receiving one dose (approximately 0.5 ml) intramuscularly. Group 9 of 30-day-old SPF chickens received 0.5 ml of sterile saline intramuscularly. 28 days post-immunization, blood was collected from each group of chickens via vein, and serum was separated to determine the titer of the egg drop syndrome virus (EDS) HI antibody in each chicken's serum.

[0072] 5. Experimental results showed that 20 days after immunization with the two batches of live vaccine, 3 / 10 of the control group developed the disease, while all chickens in the vaccine-immunized group remained normal. Pharyngeal swabs collected 5 days after challenge with the virulent virus showed that 2 out of 10 pharyngeal swabs from the untested immunization group (batch 202501) were positive, and 1 out of 10 pharyngeal swabs from the immunization group (batch 202502) was positive. All pharyngeal swabs from the control group were positive. Tracheal ciliary activity scores were all above 20 in all control groups and below 10 in the immunized group. This indicates that the live vaccine prepared from the LGX-B strain can produce a good immune protective response against virulent strains of the same lineage after immunization. Detailed results are shown in Table 13. The pharyngeal swab virus positivity rate reflects the viral shedding in the live vaccine-immunized group and the control group after challenge with the virulent virus, reflecting the protective efficiency of the vaccine.

[0073] Table 13. Immunization protection against different types of virulent strains using LGX-B strain.

[0074] Table 14. Efficacy test data of the combined live vaccine for infectious bronchitis and Newcastle disease in chickens against virulent Newcastle disease virus.

[0075] Inactivated vaccine efficacy: Tracheal samples from chickens immunized with the three batches of inactivated vaccines were tested after challenge with virulent strain I0722 / 17. Results showed that one chicken in each of batches 202503 and 202505 tested positive, while no virus was detected in batch 202504. In contrast, all samples from the control group tested positive. This indicates that the monovalent, bivalent, and trivalent inactivated vaccines prepared from the LGX-B strain can produce a good immune protective response against virulent IBV after immunization. Detailed results are shown in Table 15. The tracheal virus positivity rate data for challenged chickens reflects the residual virus in the inactivated vaccine immunized and control groups after challenge with virulent strain, reflecting the vaccine's protective effect. The bivalent and trivalent vaccines provided complete protection against Newcastle disease (ND) with a protection rate of 10 / 10 (see Table 16 for details). The average HI antibody level against egg drop syndrome virus (EDS) after immunization with the trivalent vaccine was greater than 10 log2, indicating complete protection against EDS (see Table 17 for details). Table 15. Immunogenic efficacy data of monovalent inactivated vaccine, bivalent inactivated vaccine, and trivalent inactivated vaccine against virulent strain I0722 / 17 of infectious bronchitis in chickens.

[0076] Table 16. Efficacy test data of bivalent inactivated vaccines for infectious bronchitis and Newcastle disease in chickens, and trivalent vaccines for infectious bronchitis, Newcastle disease, and egg drop syndrome against virulent Newcastle disease.

[0077] Table 17. Data on the immunogenicity of trivalent vaccines against egg drop syndrome in chickens, including those for infectious bronchitis, Newcastle disease, and egg drop syndrome.

[0078] The above results show that the active immunization protection rate of the LGX-B strain prepared into monovalent live vaccine, bivalent live vaccine, and monovalent, bivalent, and trivalent inactivated vaccines all reached over 80%, indicating that the strain of this invention has good protective efficacy against infectious bronchitis in chickens.

Claims

1. A GI-13 lineage infectious bronchitis virus in chickens ( Infectious bronchitis virus A weakly virulent strain, characterized in that, The strain was named LGX-B, a weakened strain of infectious bronchitis virus in chickens, and was deposited at the China General Microbiological Culture Collection Center (CGMCC) on November 26, 2025, with accession number CGMCC NO. 47087.

2. A microbial preparation containing the attenuated strain of GI-13 lineage chicken infectious bronchitis virus as described in claim 1 and its derivative viruses.

3. A live attenuated vaccine composition, characterized in that, The attenuated vaccine composition comprises the GI-13 lineage chicken infectious bronchitis virus attenuated strain as described in claim 1 or the microbial preparation as described in claim 2.

4. The attenuated vaccine composition according to claim 3, characterized in that, The attenuated vaccine composition is a monovalent vaccine, a bivalent vaccine, or a multivalent vaccine.

5. The inactivated vaccine composition according to claim 3, characterized in that, The attenuated vaccine composition also includes an oily adjuvant.

6. The inactivated vaccine composition according to claim 3, characterized in that, The attenuated vaccine composition also includes an immune adjuvant.

7. The attenuated vaccine composition according to claim 3, characterized in that, The viral load of infectious bronchitis virus in chickens is ≥10. 6.0 EID 50 / 0.1ml.

8. The use of the attenuated vaccine composition of claim 3 in the preparation of a drug for the prevention of infectious bronchitis in chickens.

9. The use of the GI-13 lineage attenuated strain of infectious bronchitis virus of chickens as described in claim 1 or the microbial preparation as described in claim 2 in the preparation of a vaccine for the prevention of infectious bronchitis of chickens.

10. The application according to claim 9, characterized in that, The vaccine can provide immune protection to chicken embryos or chickens after they have been immunized.

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