GI-19 pedigree chicken infectious bronchitis virus adapted to replication and proliferation of African green monkey kidney cell line and application of GI-19 pedigree chicken infectious bronchitis virus

Abstract

The invention discloses a GI-19 pedigree chicken infectious bronchitis virus adapted to replication and proliferation of an African green monkey kidney cell line and application of the GI-19 pedigree chicken infectious bronchitis virus, and belongs to the technical field of attenuated vaccines. The invention aims to provide a GI-19 pedigree IBV attenuated vaccine strain adapted to a Vero in-vitro cell line, which has good safety to both chick embryos and chickens and can stimulate effective immune protection after immunizing the chickens and the immune chick embryos. The invention provides a GI-19 pedigree avian infectious bronchitis virus attenuated strain adapted to replication and proliferation of an African green monkey kidney cell line, which is preserved in China General Microbiological Culture Collection Center on February 13, 2025, and the preservation number is CGMCC NO.46375. The invention further provides a preparation method of the GI-19 pedigree avian infectious bronchitis virus attenuated strain. The method is used for in-vitro proliferation candidate strains of viruses required by avian infectious bronchitis vaccines, diagnostic preparations and the like, and has important practical application value.

Description

Technical Field

[0001] The present invention belongs to the technical field of attenuated vaccines, and in particular relates to a GI-19 lineage chicken infectious bronchitis virus adapted to replication and proliferation in an African green monkey kidney cell line and an application thereof. Background Art

[0002] Infectious bronchitis (IB), a serious acute respiratory infection of chickens caused by the infectious bronchitis virus (IBV), is a major epidemic facing poultry industries worldwide. Since its discovery in the 1930s, IBV has caused significant economic losses to the chicken industry annually, making it a significant problem for poultry farming worldwide. This disease can cause morbidity and mortality in chickens of all ages and breeds, with extremely serious consequences, manifesting in four key ways: 1) Infection in chicks causes severe morbidity and mortality, with some strains resulting in mortality rates exceeding 80%, leading to direct economic losses; 2) Viral infection in chickens creates conditions for secondary infection with bacteria, mycoplasmas, and other pathogens, easily leading to mixed infections, resulting in higher morbidity and mortality, and increased culling rates; 3) Viral infection in broilers slows weight gain and reduces feed utilization; 4) Viral infection in laying hens causes permanent damage to the reproductive system in hens, resulting in "water sac chickens" and "false hens," and infection in adult hens leads to a decrease in egg production and quality. Therefore, IBV poses a significant threat to the poultry industry and is a critical disease issue that needs to be addressed urgently in my country and globally.

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

[0004] Vaccination is the most effective means of preventing and treating the disease. Currently commercially available IBV vaccines include attenuated live and inactivated vaccines. IBV vaccine antigens are all prepared from cultured and propagated viruses. However, the culture of infectious bronchitis virus antigens almost exclusively relies on chicken embryo production systems, rather than cell-based production systems. The reason is well known: avian coronaviruses, including infectious bronchitis virus, cannot propagate and replicate in cell lines in vitro without acclimation. Therefore, in vitro cell culture methods for IBV are rare, and in vivo culture in chicken embryos is currently the primary in vitro culture method for basic and applied research on IBV. IBV proliferates well in the allantoic cavity and chorioallantoic membrane of 9-11-day-old chicken embryos and is widely used for IBV isolation and identification, attenuation through passage, and vaccine production. However, this culture method, which requires a large number of chicken embryos, is not only expensive but also poses a risk of environmental pollution. Furthermore, the chicken embryo is a multicellular, hybrid organism, making it difficult to study IBV pathogenicity. The closed chamber environment also hinders in-depth investigation of IBV biological characteristics and virus rescue. This culture method has, to some extent, limited basic research on IBV and also affected vaccine development and production efficiency.

[0005] In vitro passage cell lines are the most economical, convenient, and stable method for culturing viruses. Furthermore, cell culture generally does not present the potential for contamination by other pathogens that may exist in chicken embryos. The cell culture components are relatively simple, and the purity of the proliferated viruses is better. In particular, the African green monkey kidney cell line (Vero) retains stable passage characteristics, and its high sensitivity to a variety of viruses, lack of interferon secretion, and the advantages of large-scale suspension culture have made it widely used in the production of human and animal coronavirus vaccines, including the new coronavirus, and in the exploration of the basic theory of viral infection. However, different coronaviruses have different adaptability to Vero cells. Compared to other coronaviruses, IBV is not easily adapted to Vero cells for culture and proliferation. Currently, only the Beaudette strain of the infectious bronchitis virus GI-1 genotype (serotype) has been widely reported to be adapted to the Vero cell line, but its immunogenicity differs significantly from that of the parental strain. While the LDT3 / 03 Vero-adapted strain retains safety for chickens and offers better protection against strains of the same lineage, it has a high lethality rate in chicken embryos and is unsafe. At the same time, the Beaudette adapted Vero cell line and the LDT3 / 03 Vero adapted strain cannot stimulate effective cross-immune protection against the dominant gene lineage strain GI-19 lineage currently prevalent in China.

[0006] Traditional vaccinations (such as nasal drops, eye drops, or drinking water) have problems such as unstable immune effects and cumbersome procedures. Therefore, in-embryo vaccination, as an emerging technology, has received widespread attention in recent years. Its technical advantages are that it improves the efficiency of antigen presentation by targeting the embryo's mucosa or immune cells (such as macrophages); chicks can obtain immunity before hatching, reducing the risk of early infection and providing early protection; automated equipment can achieve large-scale vaccination, reducing labor costs and increasing operational efficiency; it avoids the stress of multiple handling and vaccination of chicks after hatching, reducing stress; and direct delivery within the embryo ensures accurate vaccine dosage. Therefore, in-embryo vaccination has the potential to become an important tool for the prevention and control of IBV and other poultry diseases. However, currently, live IBV viruses, including commercial IBV vaccine strains, can replicate and proliferate in chicken embryos, causing damage to the embryo and making them unsuitable for use as antigens for in-embryo vaccination. For example, the QXL87 vaccine strain and the SCAU-D90 vaccine strain are currently available attenuated vaccine strains of avian infectious bronchitis virus. Both can only replicate and proliferate in chicken embryos but not in Vero cells. They also cause damage to the chicken embryos and cannot be used as antigens for in-embryo immunization. They can only be used to immunize chickens but cannot immunize the chicken embryos to produce protection.

[0007] There is an urgent need to cultivate highly cell-adapted, embryo-safe strains suitable for in-ovo immunity. In particular, there is a lack of attenuated strains for in-ovo immunity against the GI-19 lineage, the dominant strain currently circulating in China. This severely limits the development and production of vaccines for this type of infectious bronchitis virus. Therefore, screening and cultivating attenuated GI-19 lineage IBV strains adapted to Vero cell lines in vitro is a critical issue that needs to be addressed in the field of IBV research. Summary of the Invention

[0008] The purpose of the present invention is to provide a GI-19 lineage IBV attenuated vaccine strain adapted to the Vero in vitro cell line, which has good safety for both chicken embryos and chickens and can stimulate effective immune protection after immunizing chickens and chicken embryos.

[0009] The present invention provides a GI-19 lineage infectious bronchitis virus (Infectious bronchitis virus) attenuated strain adapted to replication and proliferation in an African green monkey kidney cell line. The attenuated infectious bronchitis virus strain is named I0918 / 22 Vero-adapted Strain and was deposited in the General Microbiology Center of the China Culture Collection Administration on February 13, 2025, with the deposit number being CGMCC NO.46375.

[0010] The present invention provides a microbial preparation containing the GI-19 lineage avian infectious bronchitis virus.

[0011] The present invention provides an attenuated vaccine composition / inactivated vaccine composition, which comprises the above-mentioned attenuated strain of GI-19 lineage avian infectious bronchitis virus.

[0012] It is further defined that the attenuated vaccine composition is a monovalent vaccine, a bivalent vaccine or a multivalent vaccine.

[0013] It is further defined that the inactivated vaccine composition also includes an oily adjuvant or an immune complex adjuvant.

[0014] It is further defined that the content of avian infectious bronchitis virus is ≥10 5.0 TCID 50 / 0.1ml.

[0015] The present invention provides an application of the above-mentioned GI-19 lineage avian infectious bronchitis virus attenuated strain in establishing an animal model for evaluating the effects of avian infectious bronchitis virus on cell metabolism, transcription and immunity after infecting cells.

[0016] The present invention provides an application of the attenuated vaccine composition in preparing a medicine for preventing chicken infectious bronchitis.

[0017] The present invention provides a use of the above-mentioned GI-19 lineage avian infectious bronchitis virus attenuated strain in the preparation of a vaccine for preventing avian infectious bronchitis.

[0018] It is further defined that the vaccine can be used to immunize chicken embryos or chickens to produce immune protection.

[0019] Beneficial effects: The present invention uses the typical representative strain ck / CH / LLN / 220918 of the GI-19 lineage, the dominant strain type of avian infectious bronchitis virus that has been popular in China in recent years, as the parent virus. The serotype and genotype are significantly different from the classic serotype strains used commercially in the past. Through the adaptive passage technology of chicken embryo-adapted viruses in vitro cell lines (Vero), a weak strain of GI-19 lineage avian infectious bronchitis virus that can effectively infect Vero cells and proliferate and replicate in large quantities in the cells is screened and obtained. The Vero cell-adapted infectious bronchitis virus obtained by the present invention can be used as a seed virus for preparing infectious bronchitis attenuated virus, and can be used for in vitro proliferation of candidate strains of viruses required for avian infectious bronchitis vaccines, diagnostic preparations, etc., and has important practical application value.

[0020] [Biological Deposit Information]: A GI-19 lineage infectious bronchitis virus (Infectious bronchitis virus) adapted to replicate and proliferate in African green monkey kidney cell lines. The infectious bronchitis virus of chickens is named I0918 / 22 Vero-adapted Strain and was deposited in the General Microbiology Center of the China Culture Collection of Microorganisms on February 13, 2025. The deposit number is: CGMCC NO.46375, and the deposit address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Figure 2 shows cytopathic effects of the cultured cell-adapted virus I0918 / 22 Vero-adapted Strain, the parental virulent ck / CH / LLN / 220918 strain, and the conventional vaccine strain H120 after infection of Vero cells. A shows Vero cells infected with the ck / CH / LLN / 220918 strain, B shows Vero cells infected with the H120 strain, C shows Vero cells infected with the I0918 / 22 Vero-adapted Strain, and D shows a Vero cell control.

[0022] Figure 2 Indirect immunofluorescence identification of I0918 / 22 Vero-adapted strain; A is Vero cells infected with ck / CH / LLN / 220918 strain, B is Vero cells infected with H120 strain, C is Vero cells infected with I0918 / 22 Vero-adapted strain, and D is Vero cell control;

[0023] Figure 3 This is a growth kinetics characteristic diagram of the I0918 / 22 Vero-adapted Strain, where the horizontal axis is time and the vertical axis is virus titer. DETAILED DESCRIPTION

[0024] The parent virus ck / CH / LLN / 220918 of the in vitro cell-adapted strain I0918 / 22 Vero-adapted Strain prepared by the present invention was isolated, identified and preserved by the Harbin Veterinary Research Institute of the Chinese Academy of Agricultural Sciences (unreported).

[0025] The parental virulent ck / CH / LLN / 220918 strain is GenBank: KT852992.1.

[0026] Monoclonal antibody 4F10 can be found in the literature: Zongxi Han, Fei Zhao, Yuhao Shao, Xiaoli Liu, XiangangKong, Yang Song, Shengwang Liu. Fine level epitope mapping and conservation analysis of two novel linear B-cell epitopes of the avian infectious bronchitis coronavirus nucleocapsid protein. Virus Research, 2013, 171:54–64.

[0027] The H120 strain is a GI-1 lineage commercial vaccine strain that is currently widely used in the market.

[0028] The LDT3 / 03 Vero adapted strain is a strain adapted to Vero cells that was previously cultivated and screened by the inventors. The patent number is ZL 2021 1 1487158.2 and belongs to the GI-22 lineage.

[0029] The Beaudette strain adapted to Vero cells is a recognized model virus for cell adaptation of the GI-1 lineage infectious bronchitis virus. It is the first typical IBV strain adapted to the continuous cell line Vero since its discovery and a mass-type virus of the GI-1 lineage. For details of our work on this virus, please see Dan Shan, Shouguo Fang, Zongxi Han, Hui Ai, Wenjun Zhao, Yuqiu Chen, Lei Jiang, Shengwang Liu. Effects of hypervariable regions inspike protein on pathogenicity, tropism, and serotypes of infectious bronchitis virus. Virus Research, 2018, 250:104–113.

[0030] M41 is the recognized model virus of infectious bronchitis virus and the first typical strain since the discovery of IBV. It is widely known as Mass virus (GI-1 lineage). Our article on this virus is detailed in 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.

[0031] Strain 4 / 91 is an attenuated vaccine strain of the GI-13 lineage IBV strain prevalent in Europe. It differs significantly from the strain prevalent in my country, with distinct genotypes and serotypes. For details of our research, 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.

[0032] Example 1. Obtaining GI-19 lineage chicken infectious bronchitis virus adapted to replication and proliferation in African green monkey kidney cell lines

[0033] 1. Cultivation of Vero-adapted strain (I0918 / 22 Vero-adapted Strain)

[0034] 1) Adaptation of ck / CH / LLN / 220918 on Vero cells: Vero cells were passaged according to conventional methods. After the cells grew into a monolayer, the allantoic fluid of the avian infectious bronchitis virus ck / CH / LLN / 220918, which has independent intellectual property rights, was diluted 10-fold in serum-free DMEM and inoculated into the Vero cells. The cells were incubated at 37°C for 1 hour, washed three times with PBS, and then replaced with 2% FBSDMEM growth medium. The cells were then cultured at 37°C, 5% CO2, and the cytopathic effect was observed daily. After 72 hours of culture, the virus-containing cell suspension was harvested and stored at -70°C for further passage. According to the above method, the virus was continuously passaged on Vero cells. When the virus was passaged to the P5 generation, cytopathic effect was observed under a microscope, thus obtaining the Vero-adapted strain I0918 / 22.

[0035] 2) Continuous Passaging of I0918 / 22 Vero-adapted Strain: After obtaining the Vero cell-adapted strain I0918 / 22 Vero-adapted Strain capable of infecting Vero cells and causing cytopathic effects, the virus was continuously passaged on Vero cells to passage P25 using the same passaging method as above. The Vero cell-adapted strain I0918 / 22 Vero-adapted Strain caused significant cytopathic effects upon infection, whereas the parental virus ck / CH / LLN / 220918 strain and the conventional live vaccine strain H120 did not cause cytopathic effects upon infection of Vero cells, consistent with the uninfected Vero control cells. Figure 1 ).

[0036] 3) Virus titer determination of I0918 / 22Vero-adapted Strain: The virus titer of I0918 / 22Vero-adapted Strain P25 was determined on Vero cells. The virus of I0918 / 22Vero-adapted Strain P25 was diluted 10-fold in serum-free DMEM, and 4 dilutions (10 -4 ~10 -7 ) were inoculated into Vero cell monolayers, 8 wells were inoculated with 0.1 mL per well for each dilution, and the cells were incubated at 37°C. Cell changes were observed every day, and the number of cytopathic effects was counted after 7 days. The TCID of the virus was calculated using the Reed-Muench method. 50 The results showed that the titer of I0918 / 22 Vero-adapted Strain was 10 5.0 TCID 50 / 0.1mL.

[0037] 4) Indirect immunofluorescence identification of viruses: The I0918 / 22 Vero-adapted Strain obtained by indirect immunofluorescence identification was used to inoculate Vero cell monolayers. When cytopathic effects appeared, the culture medium was discarded and the cells were fixed with 4% paraformaldehyde. An anti-avian infectious bronchitis virus monoclonal antibody (4F10) with independent intellectual property rights was used as the primary antibody, and a commercial FITC-labeled anti-mouse IgG was used as the secondary antibody. The cells were observed under a fluorescence microscope to see if specific fluorescence appeared in the cytopathic effects. At the same time, Vero cells were infected with the parent virus ck / CH / LLN / 220918 strain and the conventional live vaccine strain H120 strain, as well as normal control cells as controls, and indirect immunofluorescence was performed simultaneously. The results are shown in FIG. Figure 2 As shown, the I0918 / 22 Vero-adapted strain showed obvious specific fluorescence after infecting cells, while the parental virus ck / CH / LLN / 220918 strain and the conventional live vaccine strain H120 strain did not show obvious specific fluorescence when infecting Vero cells, which was consistent with the Vero control cells that were not infected with the virus.

[0038] II. Replication Kinetics of the I0918 / 22 Vero-adapted Strain

[0039] 1) Infection and sampling of I0918 / 22 Vero-adapted Strain: The I0918 / 22 Vero-adapted Strain virus solution was inoculated into well-grown CEF cells and Vero cells at an MOI of 0.01, incubated at 37°C for 1 h, washed three times with PBS, replaced with 2% FBSDMEM growth maintenance medium, and cultured at 37°C, 5% CO2. The virus-containing cell suspension was harvested 12 h, 24 h, 36 h, 48 h, 60 h, and 72 h after culture. At the same time, the parental strain was simultaneously infected and sampled at the 5th (P5), 10th (P10), 15th (P15), 20th (P20), and 25th (P25) passages in Vero cells and stored at -70°C for later use.

[0040] 2) Viral titer determination: The virus fluids from cell cultures harvested at different times were diluted 10-fold in serum-free DMEM. Appropriate dilutions were selected and inoculated into Vero cell monolayers. 8 wells were inoculated with 0.1 mL per well for each dilution. The cells were incubated at 37°C and the cell changes were observed daily. After 7 days, the number of cytopathic effects was counted and the TCID of the virus was calculated using the Reed-Muench method. 50 , the results are as follows Figure 3As shown, as the number of virus passages in Vero cells increases, the virus proliferation ability increases, the virus titer increases, and the time to peak value decreases. The titer of the virus propagated by the I0918 / 22 Vero-adapted strain is more stable after passage.

[0041] III. Attenuation Evaluation and Genetic Characteristics of the I0918 / 22 Vero-adapted Strain

[0042] 1) Evaluation of the attenuation of I0918 / 22 Vero-adapted Strain: 30 one-day-old SPF chickens were randomly divided into three groups (10 chickens in each group). The three groups of chickens were housed in three negative pressure isolators, with free access to feed and water. The first group of chickens was infected with I0918 / 22 Vero-adapted Strain (containing 10 5.0 TICD 50 ) were inoculated, with 100 μL per nose; the second group of chicks served as the parental virulent control group, with 100 μL ck / CH / LLN / 220918 strain (10 5.5 EID 50 ) virus solution; Group 3 chicks served as a blank control group, receiving 100 μL of cell culture medium instilled into each nasal cavity. From the date of inoculation, the incidence and mortality of the inoculated chickens were observed and recorded daily, and dead chickens were autopsied. Results: No obvious morbidity or mortality was observed in the chickens infected with the I0918 / 22 Vero-adapted Strain (Table 1). Autopsies at the end of the observation period also revealed no specific pathological changes, such as tracheal hemorrhage and kidney enlargement, consistent with the blank control SPF chickens inoculated with cell culture medium. In contrast, the SPF chicks in the parental virulent challenge group had a 100% morbidity rate and a mortality rate of 3 / 10. Affected chickens exhibited symptoms such as depression, retracted necks, arched backs, coarse and matted fur, drooping wings, open mouth breathing, and frequent head shaking. Autopsies of dead chickens revealed enlarged, whitish kidneys with urate deposits, giving them the typical "mottled kidney" appearance.

[0043] Table 1 Pathogenicity of I0918 / 22 Vero-adapted Strain to SPF chickens

[0044]

[0045] Genomic characteristics of the I0918 / 22 Vero-adapted Strain: A gene fragment of the I0918 / 22 Vero-adapted Strain was amplified by RT-PCR, cloned, and sequenced. The nucleotide and deduced amino acid sequences of the cell-adapted strain were compared with those of the parental, virulent strain, ck / CH / LLN / 220918. Genomic differences between the I0918 / 22 Vero-adapted Strain and the parental, virulent strain include varying degrees of nucleotide mutations in the 1ab, 1a, S, 3c, M, and N gene segments (G50T, G13910C, C21083A, T22547C, A22684G, T24267G, and G24291A). These genetic changes are crucial for viral adaptation and attenuation of virulence.

[0046] 2) Safety evaluation of I0918 / 22Vero-adapted strain on chicken embryos: 120 commercial chicken embryos at 18 embryonic age were randomly divided into 3 groups (40 in each group). The first group of chicken embryos were inoculated with I0918 / 22Vero-adapted strain. Each embryo was injected with 100 μL (containing 10 3.0 TICD 50 ); The second group of chicken embryos were inoculated with the parent virulent ck / CH / LLN / 220918 strain, and each embryo was injected with 100 μL (containing 10 3.0 EID 50 ) virus solution; a third group of chicken embryos served as a control, with each embryo receiving an injection of 100 μL of cell culture medium through the air chamber. After inoculation, the three groups were placed in separate incubators for continued incubation. At 20 embryonic days, the embryos were transferred to trays. At 22 embryonic days, the number of chicks born, the number of dead embryos, the number of chicks with disease, and the number of chicks alive were recorded. Dead embryos were also necropsied and tested for virus. Results: The chick hatch rate, chick survival rate, and kidney and tracheal lesion rates in the I0918 / 22 Vero-adapted Strain-infected group were not significantly different from those in the control group (Table 2). Necropsies of dead embryos in the I0918 / 22 Vero-adapted Strain-inoculated group showed no specific pathological changes, such as tracheal hemorrhage, kidney enlargement, or urate deposition, consistent with those observed in blank control embryos inoculated with cell culture medium. The hatching rate in the group inoculated with the parental strain was only 30%. Chicks hatched between one and seven days of age exhibited symptoms such as depression, retracted necks, arched backs, ragged fur, drooping wings, open-mouthed breathing, and frequent head shaking, ultimately leading to death. Autopsies of the dead chicks revealed enlarged, whitish kidneys with urate deposits, giving them a typical "mottled kidney" appearance. Two chicks each died in the group inoculated with the I0918 / 22 Vero-adapted strain and the control group, but both showed no obvious pathological changes and were presumed to be normal, weak chicks.

[0047] Table 2 Pathogenicity of I0918 / 22 Vero-adapted Strain to SPF chickens

[0048]

[0049] IV. Stability of the I0918 / 22 Vero-adapted Strain and the Characteristics and Genetic Variation of Its Derivative Viruses

[0050] 1) Subculture of the I0918 / 22 Vero-adapted strain: The I0918 / 22 Vero-adapted strain was serially passaged on Vero cells up to passage P50. Each passage of the virus produced significant cytopathic effects upon infection of Vero cells, demonstrating that the adapted virus maintained its ability to stably infect cells. Viruses from different passages were collected and stored at –70°C until further use.

[0051] 2) Virulence evaluation of progeny viruses derived from the I0918 / 22 Vero-adapted Strain: Thirty one-day-old SPF chickens were randomly divided into three groups (10 chickens in each group). The three groups of chickens were housed in three negative pressure isolators, with free access to feed and water. The chicks in groups 1 and 2 were infected with the 30th generation (10 5.5 TCID 50 ) and the 50th generation (10 5.6 TCID 50 ) were inoculated with 100 μL of cell culture medium per chick; a third group of chicks served as a blank control group, receiving 100 μL of cell culture medium per chick. From the date of inoculation, morbidity and mortality in the inoculated chicks were observed and recorded daily, and deceased chicks were autopsied. Results: No significant morbidity or mortality was observed in chicks infected with the I0918 / 22 Vero-adapted strain progeny virus. Necropsies at the end of the observation period also revealed no specific pathological changes, such as tracheal hemorrhage or renal enlargement, consistent with those observed in blank control SPF chicks inoculated with cell culture medium.

[0052] 3) Changes in the genome of the I0918 / 22 Vero-adapted Strain at different passages: The complete genome genes of the 30th and 50th passages of the I0918 / 22 Vero-adapted Strain were amplified by RT-PCR, and then gene cloning and sequencing were performed. The gene sequences of the 40th and 50th passage viruses were compared with the I0918 / 22 Vero-adapted Strain before passage. The results showed that the differences between the derived progeny viruses and the parental virulent strain included varying degrees of nucleotide mutations or deletions in the 1ab, 1a, S, 3a, 3b, 3c, M, and N gene segments. The change rate of these genes was kept within 1%, and these gene changes did not affect the virus's ability to infect Vero cells.

[0053] 4) Transmission Characteristics of Infectious Bronchitis Virus: The I0918 / 22 Vero-adapted Strain is a coronavirus, and its replication characteristics exhibit typical coronavirus characteristics. This particularity is that viral replication and proliferation rely on the virus's own RNA polymerase, which lacks proofreading capabilities. Therefore, genetic mutations during viral passage and propagation are common for this type of virus. The mutation rate of the progeny viral genomes derived from the I0918 / 22 Vero-adapted Strain of the present invention after passage is maintained within 1%, maintaining the viral replication characteristics and attenuated virulence properties.

[0054] Example 2. Evaluation of the immune efficacy of I0918 / 22 Vero-adapted strain in chicken embryos

[0055] 1) In-embryo immunization: 60 commercial chicken embryos at 18 embryonic age were randomly divided into 2 groups (30 in each group). The chicken embryos in the first group were inoculated with the I0918 / 22 Vero-adapted strain. Each embryo was injected with 100 μL (containing 10 3.0 TICD 50 ); a second group of chick embryos served as a control, each receiving 100 μL of cell culture medium injected through the air chamber. After inoculation, the two groups were placed in separate incubators for further incubation. At 20 embryonic days, the chicks were transferred to trays. Ten healthy chicks from each group were randomly selected for humoral immunity and immune challenge testing.

[0056] 2) Immune activation humoral immunity efficacy evaluation test: 2 groups of 20 one-day-old chicks were raised in separate negative pressure isolators, with free access to food and water. The first group was inoculated intra-ovarian with the I0918 / 22 Vero-adapted strain (10 3.0 EID 50Chicks from the first group (1) were immunized; chicks from the second group served as the control group. At 28 days post-immunization, serum was collected from both groups every five days for specific antibody testing using the virus-neutralizing antibody assay. The average neutralizing antibody titer reached 1 / 16, compared to 0 in the control chickens (see Table 3).

[0057] 3) Clinical protection evaluation: At 28 days of age, chicks in the in-embryo immunization group and the control group were infected with the virulent ck / CH / LLN / 220918 (10 6.5 EID 50 The chickens were challenged intranasally with 100 μL of the I0918 / 22 Vero-adapted strain (V-100 / 0.1 ml) per bird. Following challenge, the birds were observed and recorded daily for morbidity and mortality. The results are shown in Table 3. In both experimental groups, the I0918 / 22 Vero-adapted strain-immunized chickens showed no clinical symptoms and no mortality, while 6 / 10 birds in the control group became ill, including 3 deaths. This indicates that the I0918 / 22 Vero-adapted strain maintains good immunogenicity even after in ovo immunization, providing excellent protection against the virulent parental strain.

[0058] 4) Evaluation of tracheal detoxification protection in chicks: On the fifth day after the challenge in step 3) above, pharyngeal swabs were collected from both groups of chickens. Sterile physiological saline was added, and the swabs were inoculated into 9-day-old SPF chicken embryos after shaking and filtration sterilization. After 7 days of incubation, the detoxification of the pharyngeal swabs was determined based on the presence of dwarf embryos, runt embryos, and other infectious bronchitis virus-specific embryonic lesions in the embryos. The results are shown in Table 4. The pharyngeal swab detoxification rate of the chickens in the I0918 / 22 Vero-adapted Strain immunization group was 2 / 10, while the control group detoxified 10 / 10. This shows that the I0918 / 22 Vero-adapted Strain strain stimulates a good immune response after immunization and has a good immune protection effect against the parental strain.

[0059] Table 3 Evaluation of the immune efficacy of I0918 / 22 Vero-adapted Strain

[0060]

[0061] Table 4 The protective effect of I0918 / 22 Vero-adapted Strain immunized chicks on tracheal detoxification in chickens challenged with virulent toxins

[0062]

[0063] Example 3. Evaluation of the immune efficacy of the I0918 / 22 Vero-adapted strain in chickens

[0064] 1) Immune activation humoral immunity efficacy evaluation test: 20 5-day-old SPF chickens were randomly divided into 2 groups (10 in each group) and housed in negative pressure isolators. The chicks had free access to food and water. The chicks in the first group were infected with I0918 / 22 Vero-adapted Strain (10 3.0 EID 50 Chicks in the first group were immunized with 100 μL of the cell culture medium per eye and nose. A second group of chicks served as the control group, receiving 100 μL of the cell culture medium per nose. Serum was collected from each group 20 days after immunization for specific antibody testing using the virus-neutralizing antibody assay. Neutralizing antibody levels are reported in Table 5.

[0065] 2) Clinical protection evaluation: 20 days after immunization, two groups of chicks were infected with the homologous parent virulent ck / CH / LLN / 220918 (10 6.5 EID 50 The chickens were challenged intranasally with 100 μL of the virus (100 μL / 0.1 ml) per bird. Following challenge, the birds were observed and recorded daily for morbidity and mortality. The results are shown in Table 5. In both experimental groups, the chickens immunized with the I0918 / 22 Vero-adapted Strain showed no clinical symptoms or mortality, and the average neutralizing antibody titer reached 1 / 32. In the control group, 4 / 10 birds became ill, including one death; the neutralizing antibody titer in the control chickens was 0. This indicates that the I0918 / 22 Vero-adapted Strain has good immunogenicity and provides excellent protection against the virulent parental strain.

[0066] 3) Evaluation of tracheal detoxification protection in chicks: On the fifth day after the challenge in step 2) above, pharyngeal swabs were collected from both groups of chickens, sterile physiological saline was added, and the swabs were inoculated into 9-day-old SPF chicken embryos after shaking and filtration sterilization. After 7 days of incubation, the detoxification of the pharyngeal swabs was determined based on the presence of dwarf embryos, runt embryos, and other infectious bronchitis virus-specific embryonic lesions in the chicken embryos. The results are shown in Table 6. The detoxification rate of the pharyngeal swabs of the chickens in the I0918 / 22 Vero-adapted Strain immunization group was 2 / 10, while the detoxification rate in the control group was 10 / 10. This shows that the I0918 / 22 Vero-adapted Strain strain stimulates a good immune response after immunization and has a good immune protection effect against the parental strain.

[0067] Table 5 Evaluation of the immune efficacy of I0918 / 22 Vero-adapted Strain

[0068]

[0069]

[0070] Table 6 The protective effect of I0918 / 22 Vero-adapted Strain on tracheal detoxification in chickens challenged with virulent toxins

[0071]

[0072] Example 4.

[0073] 1. Preparation of I0918 / 22 Vero-adapted Strain Monovalent, Bivalent, and Multivalent Vaccines

[0074] Preparation of vaccine stock solution: Take I0918 / 22 Vero-adapted strain, dilute it 10-fold with serum-free DMEM, inoculate well-grown Vero cells, incubate at 37°C for 1 hour, wash 3 times with PBS, change to 2% DMEM growth maintenance medium, continue to culture at 37°C, 5% CO2, harvest the cell suspension containing virus 48 hours after culture, store at 2-8°C, and perform sterility test at the same time. The virus content should be ≥10 5.3 TCID 50 / 0.1ml.

[0075] Preparation of monovalent and bivalent live vaccines: (1) When preparing monovalent live vaccines, the virus culture fluid that has passed the sterility test and virus content determination is mixed and then mixed with sucrose gelatin protective agent at a ratio of 8.5:1 (virus fluid: protective agent). The freeze-dried protective agent should be kept at 40℃-50℃ (8% gelatin, 40% sucrose protective agent, sterilized at 115℃ for 40 minutes, stored at 4℃, and used up within 72 hours). The virus liquid should be shaken continuously during the addition process. After being fully mixed, it is the vaccine stock solution. The vaccine stock solution is aseptically packaged in quantitative form, quickly frozen and vacuum-dried, and sealed with a cover to prepare the monovalent live vaccine. It is stored below -15℃. (2) When preparing bivalent live vaccines, the virus culture fluid of the Newcastle disease vaccine attenuated La Sota strain that has passed the sterility test and virus content determination is mixed with the virus fluid of the I0918 / 22 Vero-adapted Strain strain and then the seedlings are prepared according to the above seedling scheme.

[0076] Preparation of inactivated vaccine: (1) When preparing the monovalent inactivated vaccine, after the sterility test and virus content test have been passed, the virus culture fluid is mixed and then inactivated by adding formaldehyde solution to a final concentration of 0.2%. Then shake thoroughly. Then, place it in a shaker at 37°C for inactivation. After 16 hours, the inactivation is complete. The inactivated virus solution is stored at 2-8°C and should not exceed 2 months. After mixing 94 parts of white oil for injection and 6 parts of Span-80, 2% aluminum stearate is added and sterilized by high pressure as the oil phase. The inactivated virus solution is added to Tween-80 at a ratio of 6% and shaken thoroughly to completely dissolve the Tween to form the water phase. Take 3 parts of the oil phase and place it in a colloid mill. Start the motor to stir, then slowly add 1 part of the water phase. Start the timer, the emulsification interval is 10, and emulsify at 4000 r / m for 8 minutes. Before the end of emulsification, add 1% thimerosal preservative to a final concentration of 1 / 10,000. The emulsified qualified vaccine is divided into sterilized vaccine bottles, covered and sealed to prepare the inactivated vaccine, which is then stored at 2-8°C. (2) When preparing the bivalent inactivated vaccine, the virus culture fluid that has been proliferated by the Newcastle disease vaccine strain and passed the sterility test, virus content and inactivation test is mixed with the virus culture fluid that has been proliferated by the I0918 / 22Vero-adapted Strain strain, and then the vaccine is prepared according to the above vaccine preparation plan. (3) When preparing the trivalent inactivated vaccine, the virus culture fluid that has been proliferated by the Newcastle disease vaccine strain and the egg drop syndrome vaccine strain and passed the sterility test, virus content and inactivation test is mixed with the virus culture fluid that has been proliferated by the I0918 / 22Vero-adapted Strain strain, and then the vaccine is prepared according to the above vaccine preparation plan.

[0077] II. Safety Verification of I0918 / 22 Vero-adapted Strain Vaccine Preparation

[0078] 1. Experimental Vaccines: A monovalent live vaccine of the I0918 / 22 Vero-adapted strain was prepared according to the live vaccine preparation method described in Example 2. Lot number 202401, 1000 doses / bottle. A bivalent live vaccine of the I0918 / 22 Vero-adapted strain and the Newcastle disease vaccine strain, La Sota, was prepared. Lot number 202402, 1000 doses / bottle. Vaccine Storage Conditions and Shelf Life: Store below -15°C for 12 months. Simultaneously, a monovalent inactivated vaccine of the I0918 / 22 Vero-adapted strain was prepared according to the inactivated vaccine preparation method described in Example 2. Lot number 202403. A bivalent inactivated vaccine of the I0918 / 22 Vero-adapted strain and the Newcastle disease vaccine strain, La Sota, was prepared. Lot number 202404. Prepare a trivalent inactivated vaccine containing the I0918 / 22 Vero-adapted strain, the Newcastle disease vaccine strain, the La Sota strain, and the egg drop syndrome vaccine strain. Lot number 202405. 250 doses per bottle. Vaccine storage conditions and shelf life: Store at -2 to 8°C for 12 months.

[0079] 2. Live Vaccine Safety Verification: Groups 1 and 2 (10 birds / group) of 5-day-old SPF chicks were vaccinated with live vaccines from batches 202401 and 202402, respectively, with each chick receiving 10 doses (approximately 0.03-0.05 ml) intranasally. Group 3 of 5-day-old SPF chicks received one drop of sterile saline intranasally. Following immunization, the clinical manifestations of each group were continuously observed, and incidence and mortality were recorded. Following the observation period, each group was necropsied for signs of infectious bronchitis, such as serous or catarrhal discharge in the trachea and sinuses of some affected chicks, or kidney swelling with the typical "pied-nephropathy" pattern.

[0080] 3. Safety verification of reversion of live vaccine: Fifteen 5-day-old SPF chickens were inoculated with the vaccine from batch 202401 (I0918 / 22 Vero-adapted Strain) via intranasal route, with a dose of 5×10 5.3 TCID 50, and placed in a negative pressure isolator for observation. Five days after inoculation, five chickens were killed, their trachea removed aseptically, ground and homogenized with PBS. After freeze-thawing three times, the mixture was centrifuged at 6000 rpm for 5 minutes, and the supernatant was sterilized by filtration through a 0.22 μm filter membrane. The filtrate was used to determine the viral content using 9-10 day old SPF chicken embryos. The next generation of chickens was then passaged, with an inoculation dose of 0.5 ml per chicken. The remaining 10 chickens were observed for 20 days after inoculation to observe for clinical symptoms and mortality. An autopsy was performed on the 20th day to observe for pathological changes. The above method was continued for another four generations of chickens, with 10 control chickens of the same age set up for each passage. The S1 protein gene sequence was determined by RT-PCR nucleic acid amplification in the last generation of chickens to verify the safety of the I0918 / 22 Vero-adapted strain in terms of virulence reversion.

[0081] 4. Inactivated Vaccine Safety Verification: Groups 1-3 (5 birds / group) of 1-2 month old SPF chickens were vaccinated with vaccines from batches 202403, 202404, and 202405, respectively. Two doses (approximately 1.0 ml) were injected intramuscularly into the chest of each chicken. Group 4 of 1-2 month old SPF chickens received 1.0 ml of sterile saline intramuscularly. Clinical manifestations of chickens in each group were continuously observed post-immunization, and morbidity and mortality were recorded. After 14 days of observation, the injection site was necropsied to assess vaccine absorption.

[0082] 5. The experimental results are shown in Table 7: (1) Live vaccine safety test: 20 days after immunization, the two live vaccine immunization groups showed no clinical symptoms of infectious bronchitis such as head shaking, listlessness, and ruffled feathers, consistent with the blank control group. During the observation period, 0 / 10 cases of disease occurred, and no kidney enlargement or "pied kidney" specific lesions were found in autopsies. All groups appeared normal. This shows that the monovalent live vaccine prepared with the I0918 / 22 Vero-adapted strain and the bivalent live vaccine prepared in combination with the Newcastle disease vaccine strain are safe after immunization of chickens.

[0083] Table 7 Safety test data of live infectious bronchitis vaccine

[0084]

[0085]

[0086] (2) The results of the safety verification of the reversion of the live vaccine are shown in Table 8: The results showed that no clinical symptoms of infectious bronchitis virus infection, such as depression, shrinking neck, arched back, rough fur, drooping wings, and open mouth breathing, were found in all the test chickens inoculated with each generation of virus within 20 days of observation. Each generation was killed on the 20th day after vaccination. No tracheal annular hemorrhage lesions were found, nor were there specific pathological changes such as "spotted kidney" and urate deposition in the kidneys. The 5th generation virus of the 202401 batch of vaccine was consistent with the I0918 / 22 Vero-adapted Strain strain virus by S1 protein gene sequencing. The above results show that the vaccine strain prepared by the I0918 / 22 Vero-adapted Strain strain will not revert to strong virulence when propagated and passaged in chickens, and its genetic characteristics are stable, making it suitable for the prevention and control of IB disease.

[0087] Table 8 Observation results of chicken passages 1 to 4

[0088]

[0089] (3) Inactivated vaccine safety test: After 14 days of observation, chickens immunized with batches 202403, 202404, and 202405 showed normal performance, consistent with the blank control group, during the observation period, with no adverse reactions. Autopsy observations at the injection site also showed no obvious inflammatory reactions such as granulomas, indicating good vaccine absorption. This indicates that both the monovalent inactivated vaccine prepared with the I0918 / 22 Vero-adapted strain, the bivalent combination 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 safe for chickens.

[0090] 3. Verification of the Immune Efficacy of the I0918 / 22 Vero-adapted Strain Vaccine

[0091] 1. Experimental Vaccines: A monovalent live vaccine of the I0918 / 22 Vero-adapted Strain strain was prepared according to the live vaccine preparation method of Example 4. Lot number 202401, 1000 doses / bottle. A bivalent live vaccine of the I0918 / 22 Vero-adapted Strain strain and the Newcastle disease vaccine strain, La Sota, was prepared. Lot number 202402, 1000 doses / bottle. Vaccine Storage Conditions and Shelf Life: Store below -15°C for 12 months. Simultaneously, a monovalent inactivated vaccine of the I0918 / 22 Vero-adapted Strain strain was prepared according to the inactivated vaccine preparation method of Example 2. Lot number 202403. A bivalent inactivated vaccine of the I0918 / 22 Vero-adapted Strain strain and the Newcastle disease vaccine strain, La Sota, was prepared. Lot number 202404. Prepare a trivalent inactivated vaccine containing the I0918 / 22 Vero-adapted strain, the Newcastle disease vaccine strain, the La Sota strain, and the egg drop syndrome vaccine strain, batch number 202405. 500 doses per bottle. Vaccine storage conditions and shelf life: Store at -2 to 8°C for 12 months.

[0092] 2. Usage and Dosage: Dilute the live vaccine according to the amount marked on the bottle label with normal saline, use a dropper to draw up the vaccine, and drip one drop (about 0.03-0.05 ml) into each chicken's nose. For the inactivated vaccine, inject 0.3-0.5 ml per chicken intramuscularly with a syringe.

[0093] 3. Live vaccine efficacy verification: Groups 1 to 2 (10 birds / group) of 5-day-old SPF chickens were vaccinated with live vaccine from batches 202401 and 202402, respectively. Each chicken was inoculated intranasally with one dose (about 0.03-0.05 ml). Group 3 5-day-old SPF chickens were each inoculated with one drop of sterile saline. 20 days after immunization, chickens in groups 1-3 were challenged intranasally with CK / CH / LLN / 220918 (10 5.5 EID 50 ) strains, 10 pharyngeal swabs were randomly collected from each group 5 days after the infection, and the clinical manifestations of the chickens in each group were continuously observed, and the incidence and mortality were recorded. The treated pharyngeal swabs were inoculated into 9-day-old SPF chicken embryos. The virus secretion of the immune and non-immune chickens after infection with the strong virus was determined based on the chicken embryo infection situation and combined with the RT-PCR method. Table 9 shows the virus positive rate. At the same time, the 4th group (10 / group) of 30-day-old SPF chicks were inoculated with the 202402 batch of live vaccine, and each chicken was inoculated with 0.01 dose (about 0.03-0.05 ml) by intranasal inoculation. Each 30-day-old SPF chicken in the 5th group was inoculated with one drop of sterile saline in the nose. 14 days after immunization, the 4-5 groups of chickens were attacked by intramuscular injection of Newcastle disease Beijing strain (10 4.0 ELD 50) strain, and the clinical manifestations of chickens in each group were continuously observed, and the incidence and mortality were recorded (Table 10) for 14 days.

[0094] 4. Inactivated vaccine efficacy verification: Groups 1 to 3 (5 birds / group) of 1-month-old SPF chickens were vaccinated with batches 202403, 202404, and 202405 of the vaccine, with one dose (about 0.5 ml) injected intramuscularly into each breast. Group 4 of 5-day-old SPF chickens were injected intramuscularly with 0.5 ml of sterile saline. 28 days after immunization, the four groups of chickens were challenged with CK / CH / LLN / 220918 (10 5.5 EID 50 ) strains, each group of chickens were killed 5 days after the infection, and the kidneys of each chicken were collected aseptically. The supernatant of the kidney tissue after homogenization was inoculated into 9-day-old SPF chicken embryos. The virus residues after the immune and non-immune chickens were infected with the strong virus were determined according to the chicken embryo infection situation and combined with the RT-PCR method. Table 11 shows the virus isolation positive rate. 5-6 groups (5 / group) of 30-day-old SPF chicks were vaccinated with 202404 and 202405 batches of inactivated vaccine, and each chicken was inoculated with 0.01 dose (about 0.03-0.05ml) by intranasal inoculation. Each 30-day-old SPF chicken in the 7th group was given a drop of sterile saline in the nose. 14 days after immunization, the 5-7 groups of chickens were attacked by intramuscular injection of Newcastle disease Beijing strain (10 4.0 ELD 50 ) strain. Clinical manifestations of chickens in each group were continuously observed, and incidence and mortality were recorded for 14 days. Eight groups (5 birds / group) of 30-day-old SPF chicks were vaccinated with the inactivated vaccine from batch 202405, with each chick receiving 0.01 dose (approximately 0.03-0.05 ml) intranasally. A ninth group of 30-day-old SPF chicks received one drop of sterile saline intranasally. Twenty-eight days after immunization, venous blood was collected from each chicken in each group, and serum was isolated and measured for the titer of EDS virus HI antibodies in each chicken's serum.

[0095] Experimental results revealed the efficacy of the live vaccine: Twenty days after immunization, two groups vaccinated with the live vaccine were challenged with the virulent CK / CH / LLN / 220918 strain. All chickens in the saline control group developed disease, exhibiting clinical symptoms of infectious bronchitis, such as head shaking, lethargy, and ruffled feathers. During the observation period, 10 / 10 chickens became ill, and 2 / 10 died. Autopsies revealed enlarged kidneys and the characteristic "mottled kidney" lesion. All chickens in the vaccine-immunized group remained normal. Throat swabs collected five days after challenge with the virulent strain tested positive for IBV in one of ten chickens from the 202402 batch, while no IBV was detected in the 202401 batch. All chickens in the control group tested positive for IBV. This demonstrates that chickens immunized with the live vaccine prepared with the I0918 / 22 Vero-adapted strain can induce a robust immune response against the homologous virulent strain. Detailed results are shown in Table 9. The virus-positive rate in the throat swabs reflects viral secretion in the live vaccine-immunized and control groups after challenge, reflecting the protective efficacy of the vaccine.

[0096] Table 9 Test data on the immune efficacy of monovalent live vaccine and bivalent live vaccine against the virulent CK / CH / LLN / 220918 strain

[0097]

[0098] Table 10 Test data on the efficacy of the bivalent live vaccine against Newcastle disease virulent NDV

[0099]

[0100] Efficacy of inactivated vaccines: 28 days after immunization, chickens in the three inactivated vaccine groups were challenged with the virulent IBV CK / CH / LLN / 220918 strain. Kidney samples were collected 5 days after challenge. One kidney sample from chickens in the 202404 batch immunization group tested positive, while no IBV was detected in the other batches. All kidney samples from the control group tested positive, demonstrating that chickens immunized with the monovalent, bivalent, and multivalent inactivated vaccines prepared from the I0918 / 22 Vero-adapted strain were able to generate a strong immune protection against the homologous virulent strain. Detailed results are shown in Table 11. The virus positivity rate in the kidneys of challenged chickens reflects the residual virus after challenge in the inactivated vaccine-immunized and control groups, reflecting the vaccine's protective efficacy. Both the bivalent and trivalent vaccines provided complete protection against Newcastle disease (see Table 12 for detailed results). The average HI antibody level against egg drop syndrome virus after immunization with the trivalent vaccine was greater than 10log2, indicating complete protection against egg drop syndrome (see Table 13 for detailed results).

[0101] Table 11 Test data on the immune efficacy of monovalent inactivated vaccine, bivalent inactivated vaccine and trivalent inactivated vaccine against ck / CH / LDL / 140520 virulent strain of chicken infectious bronchitis

[0102]

[0103] Table 12 Test data on the efficacy of the bivalent inactivated vaccine for infectious bronchitis and Newcastle disease and the trivalent vaccine for infectious bronchitis, Newcastle disease and egg drop syndrome against virulent Newcastle disease

[0104]

[0105] Table 13 Immune efficacy test of trivalent vaccine against infectious bronchitis, Newcastle disease and egg drop syndrome against egg drop syndrome

[0106]

[0107]

[0108] The results showed that the active immunization protection rate of the I0918 / 22 Vero-adapted Strain prepared into monovalent and bivalent live vaccines and monovalent, bivalent and trivalent inactivated vaccines all reached more than 80%, indicating that the strain of the present invention has good protective efficacy against avian infectious bronchitis.

[0109] Example 5. I0918 / 22 Vero-adapted Strain as a Cell Infection Model to Verify Viral Infection Mechanism

[0110] 1) Virus-infected cell model: Vero cells were cultured at a rate of 1×10 6 Plate the cells at the same number per well of a 6-well plate. For virus inoculation, sample one well, digest with trypsin, and count the cells to determine the amount of virus loaded. Wash the 6-well plate three times with PBS. Add 2 ml of serum-free medium containing 1 MOI of virus to each well. Incubate the 6-well plate at 37°C for 1 hour, discard the virus solution, wash three times with pre-chilled PBS, and switch to serum-free medium for continued incubation. The control group remains uninfected with the virus; all other procedures are the same as for the infected group. Collect infected cell samples at different time points after infection.

[0111] 2) Sample Preparation: Collect the supernatant and store in a -70°C refrigerator. To collect protein samples, wash the cells three times with pre-chilled PBS. After discarding the PBS, add 200 μl of 1× SDS protein loading buffer to each well to lyse the cells. Transfer the cells to EP tubes, boil in a water bath for 5 minutes, and centrifuge at 12,000 rpm for 5 minutes. Collect the supernatant for SDS-PAGE. For RNA extraction, 800 μl of TRIZOL reagent was added to each well of a 6-well Vero cell and CEF cell culture plate at different times after IBV infection, and the mixture was gently shaken and allowed to stand at room temperature for 3-5 minutes. After the cells were completely lysed, the lysate was transferred to an RNase-free EP tube and shaken to allow for complete lysis. 200 μl of chloroform was then added, shaken for 15 seconds, allowed to stand at room temperature for 10 minutes, and centrifuged at 12,000 rpm and 4°C for 15 minutes. The upper aqueous phase was transferred to a new RNase-free EP tube, and an equal amount of isopropanol was added and precipitated at -20°C for 1 hour; centrifuged at 12,000 rpm and 4°C for 15 minutes, the supernatant was discarded, 1 ml of 75% ethanol was added, the mixture was extracted and centrifuged at 12,000 rpm and 4°C for 15 minutes, the supernatant was discarded, and the mixture was dried.

[0112] 3) Verification of the viral entry mechanism: Vero cells were pretreated with the CME inhibitor CPZ, the Cav ME inhibitor Nystatin, and the macropinocytosis inhibitor Amiloride for 30 minutes. The cells were then infected with the I0918 / 22 Vero-adapted strain. Eight hours later, the cells were harvested and the expression of the IBV N protein was assessed by Western blot. Compared with the control pretreatment group, pretreatment with the CME inhibitor CPZ significantly reduced IBV N protein expression eight hours after infection. However, pretreatment with the Cav ME inhibitor Nystatin and the macropinocytosis inhibitor Amiloride showed no significant difference in IBV N protein expression compared with the control pretreatment group. These results demonstrate that CPZ effectively blocks IBV entry via CME, while Nystatin and Amiloride have no inhibitory effect, suggesting that IBV enters cells via CME. To confirm these results, Vero cells were pretreated with the three drugs for 30 minutes, infected with the I0918 / 22 Vero-adapted strain virus at an MOI of 1 for 2 hours, and RNA samples were collected. The inhibitory effects of the three drugs on viral genome entry were analyzed by quantitative RT-PCR. Compared with the untreated control group, CPZ pretreatment inhibited CME and significantly inhibited viral genome entry (p < 0.05), with the inhibitory effect increasing with increasing CPZ concentration. However, there was no significant reduction in viral genome entry in the Nystatin and Amiloride pretreatment groups. These results confirm that IBV relies on the clathrin-mediated endocytosis pathway for entry.

[0113] Comparative Example 1. Other strains cannot effectively adapt to proliferation on Vero cells

[0114] 1) M41 strain and 4 / 91 strain can not effectively adapt to proliferation on Vero cell: go down to posterity Vero cell according to a conventional method, after cell grows into monolayer, the allantoic fluid of Mass type avian infectious bronchitis virus typical virulent strain M41 strain and attenuated vaccine strain 4 / 91 strain is done 10 times of dilutions respectively through serum-free DMEM, inoculate Vero cell respectively, in 37 ℃ of hatching 1h, PBS is washed 3 times, changes and contains 2%DMEM growth maintenance fluid, in 37 ℃, 5%CO Condition continues to cultivate, observes cytopathic effect every day. Cultivate 72h, the results cell suspension,-70 ℃ of preservations, leave and continue to go down to posterity and use. According to aforesaid method, when blind going down to posterity to 20 generations on Vero cell continuously, can observe under the microscope, all failed to observe cytopathic effect, can not obtain Vero cell adaptation strain.

[0115] 2) Indirect immunofluorescence identification of virus: In order to confirm whether the virus replicates in Vero cells, indirect immunofluorescence identification of each generation culture was used. Vero cell cultures of M41 strain and 4 / 91 strain were blindly passaged to the 20th generation and inoculated into Vero cell monolayers. The cells were incubated at 37°C for 1 hour, washed 3 times with PBS, and then incubated in 2% DMEM for 72 hours at 37°C with 5% CO2. The culture medium was discarded and the cells were fixed with 4% paraformaldehyde. Anti-avian infectious bronchitis virus monoclonal antibody (4F10) with independent intellectual property rights was used as primary antibody. Commercial FITC-labeled anti-mouse IgG was used as secondary antibody. Observation under a fluorescence microscope indicated whether specific fluorescence occurred in the cytopathic effect. Vero cells infected with M41 strain and 4 / 91 strain and normal control cells were synchronously subjected to indirect immunofluorescence as controls.

[0116] The results showed that no specific fluorescence was observed in the Vero cells inoculated with the cultures of the M41 and 4 / 91 strains and the Vero control cells that were not infected with the virus. However, obvious specific fluorescence appeared after the cells were infected with the M41 and 4 / 91 strains, indicating that neither the traditional Mass-type typical virulent M41 strain nor the conventional attenuated vaccine strain 4 / 91 introduced from Europe could obtain a virus strain that could effectively proliferate and replicate in Vero cells through the same adaptive culture.

[0117] Comparative Example 2. Poor safety of I0918 / 22 Vero-adapted Strain parental strain and other virus strains for in-embryo inoculation

[0118] Safety evaluation of CK / CH / LLN / 220918 strain and I0918 / 22Vero-adapted P5 strain on chicken embryos: 240 commercial chicken embryos at 18 embryonic age were randomly divided into 6 groups (40 in each group). The first group of chicken embryos were inoculated with I0918 / 22Vero-adapted Strain. Each embryo was injected with 100 μL (containing 10 3.0 TICD 50 ); The second group of chicken embryos were inoculated with the parent virulent ck / CH / LLN / 220918 strain, and each embryo was injected with 100 μL (containing 10 3.0 EID 50 ) virus solution; Group 3 chicken embryos were inoculated with the parental virulent ck / CH / LLN / 220918 strain after adaptation to Vero cells, the fifth generation I0918 / 22Vero-adapted P5 strain, and each embryo was injected with 100 μL (containing 10 3.0 EID 50 ) virus solution; Group 4 chicken embryos were inoculated with the GI-1 lineage classic virulent strain M41, and each embryo was injected with 100 μL (containing 10 3.0 EID 50 ) virus solution; Group 5 chicken embryos were inoculated with GI-13 lineage attenuated strain 4 / 91, and each embryo was injected with 100 μL (containing 10 3.0 EID 50 ) virus solution, and the sixth group of chicken embryos served as a control. Each embryo was injected with 100 μL of cell culture medium through the air chamber. After inoculation, the five groups were placed in separate incubators for continued incubation. At 20 embryonic days, the embryos were transferred to trays. At 22 embryonic days, the number of hatched chicks, the number of dead embryos, the number of chicks with disease, and the number of healthy chicks were recorded. Dead chicks were also autopsied and tested for the virus. Results: The hatching rate, chick survival rate, and kidney and organ pathology rates of the chickens infected with the I0918 / 22 Vero-adapted Strain strain were not significantly different from those in the control group (see Table 14). Autopsy of the dead chicks in the I0918 / 22 Vero-adapted Strain-inoculated group revealed no specific pathological changes such as tracheal hemorrhage, kidney enlargement, and urate deposition, consistent with those observed in blank control chick embryos inoculated with cell culture medium. The hatching rate of the parental virulent inoculation group, as well as the fifth-generation Vero cell-adapted I0918 / 22 Vero-adapted P5 strain, M41 strain, and 4 / 91 strain, was less than 50%. Hatched chicks exhibited varying degrees of morbidity or mortality between days 1 and 7. This suggests that the parental strain of the I0918 / 22 Vero-adapted Strain and other viral strains exhibit poor safety for in ovo inoculation, while the I0918 / 22 Vero-adapted Strain exhibits minimal pathogenicity to chicken embryos and maintains good safety.

[0119] Table 14 Safety of different strains after inoculation into SPF chicken embryos

[0120]

[0121] Comparative Example 3. Comparative evaluation of the immune efficacy of viruses of different adaptation generations

[0122] 1) Evaluation of humoral immune efficacy of immune activation: 30 5-day-old SPF chickens were randomly divided into 3 groups (10 chickens in each group) and housed in negative pressure isolators. The chicks had free access to food and water. The chicks in group 1 were infected with I0918 / 22 Vero-adapted Strain (10 3.0 EID 50 ) were immunized, with 100 μL injected into each eye and nose; the second group of chicks used the fifth generation I0918 / 22 Vero-adapted P5 strain (10 3.0 EID 50 ) were immunized, with 100 μL injected into each eye and nose; the third group of chicks used the 50th generation I0918 / 22 Vero-adapted P50 strain (10 3.0 EID 50 Chicks in Group 4 were immunized with 100 μL of the cell culture medium per eye and nose. A fourth group of chicks served as a control group, receiving 100 μL of the cell culture medium per nose. Serum was collected from each group 20 days after immunization for specific antibody testing using the same method as for virus-neutralizing antibody testing.

[0123] 2) Evaluation of replication ability in chicken trachea: On the 5th day after immunization in step 1), pharyngeal swabs were collected from 3 groups of chickens, sterile physiological saline was added, and 9-day-old SPF chicken embryos were inoculated after shaking and filtration sterilization. After 7 days of culture, the pharyngeal swab detoxification was determined based on the presence of dwarf embryos, short embryos, and other infectious bronchitis virus-specific chicken embryo pathological changes in the chicken embryos. The results are shown in Table 15. The pharyngeal swab detoxification of the chickens in the immunized groups of I0918 / 22Vero-adapted Strain and the 50th generation I0918 / 22Vero-adapted P50 strain was 10 / 10, while the 50th generation I0918 / 22Vero-adapted P50 strain and the control group had a 0 / 10 detoxification. It can be seen that the I0918 / 22Vero-adapted Strain strain and the 5th generation I0918 / 22Vero-adapted P50 strain had a 0 / 10 detoxification. The P5 strain has the ability to replicate in the chicken respiratory tract, while the 50th generation I0918 / 22 Vero-adapted P50 strain, which is highly adapted to Vero cells, cannot proliferate and replicate in the chicken respiratory tract.

[0124] 3) Clinical protection evaluation: 20 days after immunization, 4 groups of chicks were infected with the homologous parent virulent ck / CH / LLN / 220918 (10 6.5 EID 50 The chickens were challenged with 100 μL of nasal drops of 100 μL / 0.1 ml of the virus (100 μL per bird). After the challenge, the incidence and mortality of the chickens were observed and recorded daily. The results are shown in Table 16. Among the four experimental chicken groups, the chickens in the I0918 / 22 Vero-adapted Strain and the fifth-generation I0918 / 22 Vero-adapted P5 groups showed no clinical symptoms and no mortality, and the average neutralizing antibody titer reached 1 / 32. In contrast, the chickens in the 50th-generation I0918 / 22 Vero-adapted P50 group and the control group developed disease in 3 / 10-4 / 10 birds, including one mortality each; the neutralizing antibody titer was 0. This shows that the I0918 / 22 Vero-adapted Strain and the fifth-generation I0918 / 22 Vero-adapted P5 strain have good immunogenicity and provide excellent protection against the parental virulent virus.

[0125] 4) Evaluation of tracheal detoxification protection in immunized chickens: On the fifth day after the challenge in step 3) above, pharyngeal swabs were collected from the four groups of chickens. Sterile physiological saline was added, and the swabs were inoculated with 9-day-old SPF chicken embryos after shaking and filtration sterilization. After 7 days of incubation, the pharyngeal swab detoxification was determined based on the presence of dwarf embryos, runt embryos, and other infectious bronchitis virus-specific embryo pathological changes in the chicken embryos. The results are shown in Table 17. The pharyngeal swab detoxification rate of the chickens in the I0918 / 22 Vero-adapted Strain immunization group was 2 / 10, while the control group detoxified 10 / 10.

[0126] Table 15 Results of tracheal proliferation virus excretion test after different generations of virus immunization chickens

[0127]

[0128] Table 16 Evaluation results of immune efficacy of viruses of different generations

[0129]

[0130] Table 17 The protective effect of different generations of virus-immunized chicks on tracheal detoxification in chickens challenged with strong toxins

[0131]

[0132] It can be seen that the I0918 / 22Vero-adapted Strain and the 5th generation I0918 / 22Vero-adapted P5 strain stimulated a good immune response after immunization and had a good immune protection effect against the parental strain, while the 50th generation I0918 / 22Vero-adapted P50 strain lost the ability to replicate in the chicken trachea and also lost the ability to stimulate immunity.

Claims

1. An attenuated strain of GI-19 lineage infectious bronchitis virus (IBDV) adapted to replicate and proliferate in African green monkey kidney cell lines, characterized in that: The attenuated strain of avian infectious bronchitis virus is named I0918 / 22 Vero-adapted Strain, and was deposited in the General Microbiology Center of China Culture Collection Administration on February 13, 2025, with the deposit number: CGMCC NO.46375.

2. A microbial preparation containing the GI-19 lineage avian infectious bronchitis virus according to claim 1.

3. A vaccine composition, characterized in that The vaccine composition comprises the attenuated strain of the GI-19 lineage infectious bronchitis virus of claim 1 or the microbial preparation of claim 2.

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

5. The vaccine composition according to claim 3, characterized in that The vaccine composition further comprises an oily adjuvant or an immune complex adjuvant.

6. The vaccine composition according to claim 3, characterized in that The content of avian infectious bronchitis virus is ≥10 5.0 TCID 50 / 0.1ml.

7. Use of the attenuated GI-19 lineage infectious bronchitis virus strain according to claim 1 or the microbial preparation according to claim 2 in establishing an animal model for evaluating the effects of infectious bronchitis virus on cell metabolism, transcription, and immunity after infection of cells.

8. Use of the attenuated vaccine composition according to claim 3 in the preparation of a medicament for preventing avian infectious bronchitis.

9. Use of the attenuated strain of GI-19 lineage avian infectious bronchitis virus according to claim 1 or the microbial preparation according to claim 2 in the preparation of a vaccine for preventing avian infectious bronchitis.

10. The use according to claim 9, characterized in that The vaccine can produce immune protection after immunizing chicken embryos or chickens.

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