G1-19 type chicken infectious bronchitis virus attenuated vaccine strain and application thereof

Abstract

The application discloses a GI-19 type chicken infectious bronchitis virus attenuated vaccine strain and application thereof, relates to the technical field of biology, and the GI-19 type chicken infectious bronchitis virus attenuated vaccine strain is a chicken infectious bronchitis virus SCAU-D90 strain, is preserved in the China Center for Type Culture Collection, has a preservation number of CCTCC NO:V202450, and is located in Wuhan, Wuhan University, China. The chicken infectious bronchitis virus SCAU-D90 strain is obtained by attenuating the GI-19 type chicken infectious bronchitis virus SCAU-D5 strain through 90 generations of chicken embryo passage, is safe and effective, can protect the chicken from the attack of the GI-19 type chicken infectious bronchitis virus virulent virus, has wide practical value, and effectively solves the problems that existing vaccine strains cannot effectively prevent and treat the attack of the GI-19 type chicken infectious bronchitis virus epidemic strain and have unsatisfactory prevention and treatment effects.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, and particularly relates to a GI-19 type infectious bronchitis virus attenuated vaccine strain and application thereof. BACKGROUND

[0002] Infectious bronchitis (IB) is an acute, highly contagious respiratory disease of chickens caused by infectious bronchitis virus (IBV). IBV can infect all ages and most types of chickens, especially 1-5 week old chicks are very susceptible. The main symptoms of sick chickens are respiratory symptoms, kidney enlargement, pale, and a large amount of uric acid deposition, even appearing as "spotted kidney" and causing death. For laying hens, IBV causes not only the above symptoms, but also a decrease in egg production and egg quality. Infection of young female chickens can cause permanent atrophy of the oviduct, and some IBV strains can also cause intestinal, gastric, and muscle lesions. For broilers, IBV can also cause growth retardation, weight gain and feed reward reduction in chicks, and is prone to cause mycoplasma mixed infection and secondary infection of Escherichia coli, thereby increasing the mortality rate of chicken flocks. IBV is widespread and persistent in poultry raising areas around the world, causing huge economic losses to the global poultry industry and hindering the development of the poultry industry. Therefore, IB is listed as a B-class prevention and control disease of poultry by the World Organization for Animal Health, and is listed as a class II prevention and control disease in China, and is one of the major infectious diseases that seriously affect the world's poultry industry.

[0003] IBV belongs to the order Nidovirales, the family Coronaviridae, the genus Coronavirus, and is a representative strain of the third group of avian coronaviruses. It is an RNA virus with strong mutation and recombination ability. Under the double pressure of natural environmental selection and vaccine immunity, the frequency of gene mutation of IBV is increased, which leads to the continuous emergence of new variants of IBV, resulting in the existence of 36 genetic lineages of IBV strains and more than 30 serotypes in the world. New serotypes, genotypes and variants are still emerging, and the cross-protection between different serotypes and genotypes is very weak, which brings great difficulties to the diagnosis, prevention and treatment of IB. Through epidemiological investigation and evolutionary analysis of the S1 gene (immunogenicity determining protein gene), we found that there are at least 11 lineages (genotypes) of IBV epidemic strains in China at present, and the main epidemic strain lineages include GI-19 (QX) type, GI-22 (HN08) type, GI-7 (TW) type, GI-13 (4 / 91) type and GVI-1 (TC07-2) type. The GI-19 (QX) type isolate accounts for about 70% of the number of isolates, and is the most dominant genotype epidemic strain.

[0004] Vaccines play a very important role in the prevention of IBV, at present, the vaccines used in China mainly include inactivated vaccine and attenuated vaccine. The inactivated vaccine is mainly used before the onset of breeding hens and laying hens. However, there are still many problems such as large amount of inactivated vaccine, the need to cooperate with adjuvant, complex preparation, high cost and the like. In addition, the inactivated vaccine cannot cause mucosal immunity and cellular immunity, and therefore cannot provide effective protection. The attenuated vaccine is prepared by continuous passage of a domesticated strain through chicken embryos. The attenuated vaccine can effectively stimulate the immune system of the body, can simultaneously stimulate cellular immunity, mucosal immunity and humoral immunity, is convenient to use, and has low production cost. The attenuated vaccine is widely used for immunization of broilers, breeding hens and laying hens. The IB attenuated vaccine has been used in the Netherlands since 1956, and H52 and H120 are the most widely used live vaccines in the world. Although the Mass type vaccine (H120, H52) has been widely used in China, the existing vaccine cannot provide effective protection because the similarity of the S1 gene of the vaccine strain and the currently prevalent strain in China is low (70-85%), and therefore IB has frequently and widely broken out in China in recent years.

[0005] Evolutionary analysis of the S1 gene of the IBV isolated strain in China from 2008 to 2022 shows that the GI-19 type isolated strain IBV has become the most predominant genotype of IBV in China, accounting for about 70% of the isolated strains. The similarity of the S1 gene of the GI-19 type and the commonly used vaccine strains (H120, H52, LDT3A and the like) in China is low (71-82%), and the evolutionary distance is far. Further research shows that the result of challenge with the GI-19 type strain after immunization of a chicken population with the main attenuated vaccine (H120, H52, LDT3A and the like) in China shows that the commercial vaccine strain cannot provide effective protection against the currently predominant epidemic strain. SUMMARY

[0006] In view of the above problems in the prior art, the present application provides a GI-19 type chicken infectious bronchitis virus attenuated vaccine strain and application thereof. The GI-19 type chicken infectious bronchitis virus attenuated vaccine strain is the chicken infectious bronchitis virus SCAU-D90 strain, which is safe and effective when prepared into a vaccine, can protect against challenge of the GI-19 type chicken infectious bronchitis virus strong virus, and effectively solves the problems that the existing vaccine strain cannot effectively prevent and treat the attack of the GI-19 type chicken infectious bronchitis virus epidemic strain and the problem of unsatisfactory prevention and treatment effect.

[0007] To achieve the above object, the technical scheme adopted by the present application to solve its technical problems is to provide a GI-19 type infectious bronchitis virus attenuated vaccine strain, which is the infectious bronchitis virus SCAU-D90 strain (Infectious bronchitis virus SCAU-D90), preserved in the China Center for Type Culture Collection on April 28, 2024, with the preservation number of CCTCC NO: V202450 and the preservation address of Wuhan University, Wuhan, China.

[0008] Further, the whole genome sequence of the infectious bronchitis virus SCAU-D90 strain is shown in SEQ ID NO: 1.

[0009] Further, the infectious bronchitis virus SCAU-D90 strain is obtained by attenuating the GI-19 type infectious bronchitis virus SCAU-D5 strain through 90 passages of chicken embryos.

[0010] Further, the acquisition method of the infectious bronchitis virus SCAU-D90 strain comprises the following steps: inoculating the IBV SCAU-D5 strain into 10-day-old SPF chicken embryos through the allantoic cavity, inoculating 200 μL per embryo, inoculating 5 chicken embryos, placing them in a 37℃ incubator for 36-48 h, then aseptically collecting the allantoic fluid of the chicken embryos, and then performing the next passage, and continuously passing for 90 passages according to this method, and naming it as SCAU-D90.

[0011] Further, the nucleotide sequence of the S protein encoded by the infectious bronchitis virus SCAU-D90 strain is shown in SEQ ID NO: 2, and the amino acid sequence of the S protein is shown in SEQ ID NO: 3.

[0012] Further, the nucleotide sequence of the N protein encoded by the infectious bronchitis virus SCAU-D90 strain is shown in SEQ ID NO: 4, and the amino acid sequence of the N protein is shown in SEQ ID NO: 5.

[0013] A vaccine for preventing and treating GI-19 type infectious bronchitis comprises the above infectious bronchitis virus SCAU-D90 strain and a pharmaceutically acceptable immune enhancer and a freeze-drying protective agent.

[0014] The above infectious bronchitis virus SCAU-D90 strain is used for preparing a medicine for preventing or treating infectious bronchitis of chickens.

[0015] The present application has the following beneficial effects:

[0016] 1. The application provides a GI-19 type chicken infectious bronchitis virus attenuated vaccine strain, that is, the chicken infectious bronchitis virus SCAU-D90 strain, which is preserved in the China Center for Type Culture Collection on April 28, 2024, and the preservation number is CCTCC NO: V202450, and the preservation address is: China, Wuhan, Wuhan University; the attenuated strain provided by the application is safe for chickens of various ages and various types, and has no side effects; the vaccine made of the attenuated strain is safe and effective, can protect against homologous strong virus attack, and has practical and extensive application value. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 It is a S1 gene phylogenetic analysis diagram of SCAU-D5 strain;

[0018] Figure 2 It is a survival curve diagram of 14-day-old SPF chickens inoculated with different doses of SCAU-D5 strain;

[0019] Figure 3 It is a survival curve diagram of 14-day-old SPF chickens inoculated with different generations of passage viruses;

[0020] Figure 4 It is a survival curve diagram of two GI-19 type epidemic strong viruses after immunization with SCAU-D90;

[0021] Figure 5 It is an immunoprotection efficacy dissection diagram of strong virus JL21 strain after immunization with SCAU-D90;

[0022] Figure 6 It is a dissection diagram after strong virus JL21 strain attack;

[0023] Figure 7 It is an immunoprotection efficacy dissection diagram of strong virus FJ22 strain after immunization with SCAU-D90;

[0024] Figure 8 It is a dissection diagram after strong virus JL22 strain attack. DETAILED DESCRIPTION

[0025] The principles and characteristics of the application are described below, and the examples are only used to explain the application and not to limit the scope of the application.

[0026] Example 1 Infectious bronchitis virus SCAU-D5 strain isolation and identification

[0027] SCAU-D5 strain was isolated and identified by the laboratory in 2009 from 45-day-old diseased Qingyuan broilers in Guangdong, and the specific process is as follows:

[0028] (1) Isolation and culture of virus The sterile collected dead chicken samples (kidney, spleen, lung, trachea) were cut into pieces, added into PBS containing double antibodies at a ratio of 1:5, ground thoroughly, and then repeated freeze-thawing three times. The supernatant was centrifuged at 12000 rpm for 10 min, filtered by a 0.22 μM filter, and then inoculated into 10 10-day-old SPF chicken embryos through the allantoic cavity at 0.2 mL per embryo. The embryos were incubated at 37°C in a biochemical incubator. The dead embryos were discarded at 24 h, and 5 embryos were collected at 48 h. The remaining embryos were continuously incubated until 144 h, and the embryo pathological changes were recorded. The harvested virus was stored at -80°C. The virus was blindly transmitted to the third generation of chicken embryos, which showed typical transmission virus lesions, such as delayed embryo development, embryo body curling and dehydration, etc. The virus was blindly transmitted to the fifth generation, and the allantoic fluid was harvested and named SCAU-D5, which was stored at -80°C. The allantoic fluid was determined to have a virus content of 10 5.3 EID 50 / 0.1 mL.

[0029] (2) Virus identification

[0030] 1. Hemagglutination: The allantoic fluid harvested from the 1st to 5th generations was subjected to hemagglutination test. The results showed that the allantoic fluid of each generation did not agglutinate 1% chicken red blood cells, and the virus had no hemagglutination, which excluded the possibility of being a positive myxovirus, paramyxovirus, adenovirus and other hemagglutinating avian viruses.

[0031] 2. RT-PCR identification: The allantoic fluid of SCAU-D5 was extracted with Axyprep body fluid virus DNA / RNA small amount extraction kit, and subjected to RT-PCR amplification with N gene primers.

[0032] The upstream primer N-F was 5'-CGGAGCAATAGCAAGAAAAGC-3' (SEQ ID NO: 6), and the downstream primer N-R was 5'-GCAGCAACCCACACTATACCATC-3' (SEQ ID NO: 7).

[0033] The PCR product was subjected to 1.5% agarose gel electrophoresis, and the results showed that there was a target band at about 500 bp, which proved that the isolated virus was IBV.

[0034] 3. Sequencing and analysis of transmission S1 gene

[0035] The allantoic fluid of SCAU-D5 was extracted with Axyprep body fluid virus DNA / RNA small amount extraction kit, and subjected to RT-PCR amplification with S1 gene primers.

[0036] Upstream primer S1-F: 5'-AAGACTGAACAAAAGACCGACT-3' (SEQ ID NO: 8); downstream primer S1-R: 5'-CAAAACCTGCCATAACTAACATA-3' (SEQ ID NO: 9).

[0037] The PCR product was subjected to 1.5 wt% agarose gel electrophoresis, the product length was about 1700 bp, the target band was clear without impurity band, and was directly sent to Huada Gene Company for sequencing. After splicing and arranging the S1 gene sequence, the full length was 1620 bp.

[0038] The S1 gene sequence of SCAU-D5 strain was subjected to multiple alignment with the S1 genes of currently commonly used vaccine strains H120 (EU822341), H52 (AF352315), W93 (AY842862), M41 (DQ664534), 4 / 91 (JN192154), and LDT3-A (AY702975) vaccine strains, and the homology was low (all less than 84%), being 77.7%, 78.1%, 77.4%, 76.8%, 76.6%, and 83.8%, respectively, and the homology with GI-19 type strains (QXIBV, LX4, FJ21, and JL21) was high (96.7%-99.6%). The phylogenetic analysis diagram is shown in Figure 1 .

[0039] It can be known from Figure 1 that the S1 gene of SCAU-D5 strain clusters with the S1 gene of GI-19 type strain, indicating that SCAU-D5 strain is a GI-19 type IBV isolated strain. The spike protein encoded by S1 gene is located on the surface of virus, induces the body to produce neutralizing antibody, and is closely related to the protection reaction of the body. S1 gene is the most likely to mutate during the evolution of virus, and a difference of more than 5% in the sequence may not produce or completely produce cross protection.

[0040] 4. Morphological observation

[0041] After centrifugation and phosphotungstic acid negative staining, the coronavirus with a diameter of about 80-120 nm was observed under an electron microscope, the virus particles were polymorphic, most of which were circular, had a capsule, and the surface had loose and uniform arranged crown-like protrusions. The virus had a typical morphology of coronavirus.

[0042] 5. Animal regression test

[0043] 14-day-old SPF chickens 40 were randomly divided into 4 groups, 1-3 groups were inoculated with SCAU-D5 strain virus 10 4.0 EID 50 / each, 10 5.0 EID 50 / each and 106.0 EID 50 / , the blank group was not vaccinated. After vaccination, the observation was continued for 14 days, and the results are shown in Table 1 and Figure 2 shown.

[0044] Table 1 Test results of SPF chickens inoculated with different doses

[0045]

[0046] From Table 1 and Figure 2 It can be seen that when SPF chickens were inoculated with different doses, clinical symptoms began to appear 3-4 days after the infection, and chickens began to die 4-5 days later, with an incidence rate of 80-100% and a mortality rate of 20-40%. The sick chickens showed respiratory symptoms, tracheal rales, lethargy, diarrhea, increased water intake, and decreased feed intake. The dead chickens were extremely emaciated, with slight tracheal mucus and bleeding, air sac inflammation, and significantly enlarged and mottled kidneys with a large amount of white urate deposits. After the 10th day, the clinical symptoms gradually improved, and 14 days after the infection, the surviving chickens basically returned to normal. The results showed that the isolated virus can be used to replicate clinical cases, and the inoculation dose of 10 4.0 EID 50 / It can only cause 100% disease and can be used as a counter-toxic dose.

[0047] 6. Serum neutralization test

[0048] Preparation of SCAU-D5 positive serum: Five 40-day-old SPF chickens were housed in an isolator with free access to food and water. SCAU-D5 inactivated oil-emulsion vaccine was administered subcutaneously in the neck. Immunizations were repeated three times, every two weeks, with 1 mL per bird administered each time. On day 14 after the three vaccinations, venous blood was collected from each chicken, and serum was separated and assayed for antibody levels using the IDEXX IBV Antibody ELISA Kit. Blood was collected from chickens that tested positive for antibodies with titers ≥2000, and high-immunity single-factor serum was isolated and stored at -80°C.

[0049] Cross-neutralization test with positive sera of similar diseases: SCAU-D5 strain was diluted 1000 times and mixed with equal amounts of Newcastle disease virus (NDV), infectious bursal disease virus (IBDV), infectious laryngotracheitis virus (ITLV), IBV (M41 strain) positive sera (purchased from China Veterinary Drug Administration) and SCAU-D5 strain positive sera, reacted at 37°C for 60 minutes, and inoculated into 5 10-day-old SPF chicken embryos, 0.2 mL per embryo into the allantoic cavity. The virus control group was 10 SCAU-D5 strain. -4Diluted virus was mixed with equal amount of saline. The chicken embryos were incubated at 37°C, and dissected after 144 hours. The embryos of the chicken embryos that died within 24-144 hours and the live embryos at 144 hours were observed for specific lesions such as dehydration, curling, small development (embryo weight less than 5g of the blank group) and the like. The embryos with specific lesions were taken as the infection index of IBV specific lesions to determine serum neutralization. The results are shown in Table 2.

[0050] Table 2 Results of serum neutralization test of other pathogen positive serum

[0051] Group Chicken embryo lesions (average embryo weight) Determination D5 + NDV serum 5 / 5 stunted (12.2 g) Not neutralized D5 + IBDV serum 1 / 5 dead, 4 / 5 stunted (13.3 g) Not neutralized D5 + ILTV serum 2 / 5 dead, 3 / 5 stunted (13.5 g) Not neutralized D5 + IBV (M41) serum 2 / 5 normal (18.9 g), 3 / 5 stunted (13.9 g) Incomplete neutralization D5 + IBV (D5) serum 5 / 5 normal (19.0 g) Complete neutralization D5 virus control 2 / 5 dead, 3 / 5 stunted (12.9 g) All infected Blank group 5 / 5 normal (19.7 g) Normal development

[0052] As shown in Table 2, SCAU-D5 strain cannot be neutralized by NDV, IBDV, ITLV, and cannot be completely neutralized by IBV (M41 strain) positive serum, but can be completely neutralized by IBV (M41 strain) positive serum.

[0053] Neutralization test with IB positive serum: D5 strain was diluted with normal saline to contain 100 EID50 per 0.1 mL; IBV M41 strain, 4 / 91 strain, LDT3-A strain and D5 strain positive serum were diluted by 16 times respectively, and the diluted virus and serum were mixed in equal amount, incubated at 37°C for 60 minutes, and then inoculated into SPF chicken embryos through allantoic cavity, 5 embryos per serum and 0.2 mL per embryo. The chicken embryos were incubated at 37°C, and dissected after 144 hours. The embryos of the chicken embryos that died within 24-144 hours and the live embryos at 144 hours were observed for specific lesions such as dehydration, curling, small development (embryo weight less than 5g of the blank group) and the like. The embryos with specific lesions were taken as the infection index of IB specific lesions to determine serum neutralization. The results are shown in Table 3.

[0054] Table 3 Results of IB positive serum neutralization test

[0055]

[0056] As shown in Table 3, SCAU-D5 strain is different from commonly used IBV vaccine strains M41 strain, 4 / 91 strain and LDT3-A strain in serotype, and cannot be completely neutralized.

[0057] A GI-19 type avian infectious bronchitis virus SCAU-D5 strain (D5 for short) was obtained through isolation and identification of virus, PCR identification, morphological observation, serum chicken embryo neutralization test, animal regression experiment and other pathogenic biological characteristics research.

[0058] Example 2 Cultivation of SCAU-D90 attenuated strain

[0059] 1. Attenuation of strain chicken embryo continuous passage

[0060] The D5 strain was continuously passaged using 9-11 day old SPF chicken embryos. Each passage was inoculated with 10 embryos via allantoic cavity, 0.2 mL / embryo, incubated at 37°C, and the dead embryos within 24 hours were discarded. Five live embryos were harvested at 36-48 hours, and the embryo fluid was collected aseptically for the next passage. The remaining embryos were observed for 144 hours to determine whether they had transmitted the disease. This process was repeated until the 120th passage. During the process, the virus content, sterility and mycoplasma detection, foreign virus detection, and safety test on susceptible chicks were performed at certain intervals to determine whether the virulence was attenuated.

[0061] 2. Virus content determination

[0062] The EID50 of the D5, D25, D40, D58, D75, D90, and D110 strains was determined during the passage process. The virus was diluted 10 times with sterilized physiological saline, and five dilutions (10 -4 -10 -8 ) were selected for inoculation of five 10-day-old SPF chicken embryos each, at 0.2 mL per embryo, and incubated at 37°C for 144 hours. The dead embryos within 24 hours were not counted, the number of infected embryos was recorded, and the EID50 was calculated according to the Reed-Muench method. 50 The results are shown in Table 4.

[0063] Table 4 Comparison of titers of different passages of SCAU-D5

[0064] Generation (room temperature) D5 D20 D40 D60 D80 D90 D100 EID50 / 0.2 mL 10 6.2 ]] 10 6.5 ]] 10 6.8 ]] 10 7.2 ]] 10 7.5 ]] 10 7.5 ]] 10 7.6 ]]

[0065] As shown in Table 4, the virus titer of the strain SCAU-D5 increased continuously after the passage, and reached more than 10 7.2 EID 50 / 0.2 mL after the 60th passage.

[0066] 3. Sterility, mycoplasma, and foreign virus detection During the passage process, the D5, D20, D40, D60, D80, D90, and D100 strains were subjected to sterility, mycoplasma, and foreign virus detection according to the current Appendix of the Veterinary Pharmacopoeia of the People's Republic of China. The results of each passage were negative.

[0067] 4. Safety test on susceptible chicks

[0068] During the continuous passage process, the safety test was performed on the D5, D20, D40, D60, D80, D90, and D100 strains. One-day-old SPF chicks were randomly divided into groups, with 10 chicks in each group. The test group was inoculated with each passage virus via nasal instillation and eye drops, 10 4.0 EID 50 / 100 μL, another group as control without treatment. Raising in negative pressure isolator, free drinking water and feeding. After inoculation, observing daily, recording the morbidity and mortality of chicken, and timely necropsy of dead chickens. After 2 weeks, necropsy of surviving chickens, recording the lesions of kidney, trachea and other organs. The results are shown in Table 5 and Figure 3

[0069] Table 5 Pathogenicity evaluation of different passages of virus to 1-day-old SPF chickens

[0070] Grouping Generation Morbidity Mortality 1 D5 10 / 10 4 / 10 2 D20 10 / 10 3 / 10 3 D40 10 / 10 3 / 10 4 D60 6 / 10 1 / 10 5 D80 0 / 10 0 / 10 6 D90 0 / 10 0 / 10 7 D100 0 / 10 0 / 10 8 Blank control 0 / 10 0 / 10

[0071] As shown in Table 5 and Figure 3 , after 20 passages, the pathogenicity of the passage virus to chickens gradually decreased. The morbidity of chickens inoculated with D20, D40 and D60 viruses was 100% (10 / 10), 100% (10 / 10) and 60% (6 / 10), and the mortality was 30% (3 / 10), 30% (3 / 10) and 10% (1 / 10), respectively. The viruses of D80, D90 and D100 passages had no pathogenicity, and the inoculated chickens were completely normal in spirit, without respiratory symptoms and death, and no lesions in trachea and kidney were found in necropsy.

[0072] 5. Immunogenicity test

[0073] The weakened viruses of D80, D90 and D100 passages were determined for immunogenicity. 1-day-old SPF chickens were randomly divided into 5 groups, 15 chickens in each group, and the test groups were inoculated with D80, D90 and D100, respectively, at a dose of 10 4.0 EID 50 / each. The blank control group and the challenge control group were not treated. Raising in negative pressure isolator, free drinking water and feeding. After inoculation, observing daily, recording the morbidity and mortality of chicken, and timely necropsy of dead chickens. After 2 weeks, necropsy of surviving chickens, recording the lesions of kidney, trachea and other organs. The results are shown in Table 5 and 4.0 EID 50

[0074] Table 6 Comparison of immune protection rates of different strains at different doses

[0075]

[0076] ​​From Table 6, it can be seen that the chickens in the experimental groups and the control group were normal after immunization. The challenge control group showed respiratory symptoms on the 3rd day after challenge, some chickens were listless, and the morbidity rate was 100%, 3 chickens died, and the surviving chickens had varying degrees of tracheal bleeding, tracheal mucus, and kidney enlargement and pallor. The D80 and D90 immunization groups had no morbidity and no deaths; the D100 immunization group had 20% morbidity. Considering safety and immunogenicity, the D90 virus was selected as the original seed virus of the attenuated vaccine, and the attenuated strain was named SCAU-D90 strain.

[0077] 6. Immune protection test of attenuated SCAU-D90 strain against two epidemic strains

[0078] 1-day-old SPF chicks were randomly divided into 5 groups, 15 in each group, two test groups were inoculated with SCAU-D90 strain, the inoculation dose was 10 4.0 EID 50 / each, two challenge control groups and the blank control group were not treated. They were raised in a negative pressure isolator and fed freely. After 2 weeks, one experimental group and one challenge control group were challenged with the virulent JL21 strain of GI-19 type by nasal drop, 10 4.0 EID 50 / each, another experimental group and another challenge control group were challenged with the virulent FJ22 strain of GI-19 type by nasal drop, 10 4.0 EID 50 / each, the morbidity and mortality of the chicken population were observed and recorded daily after challenge, and the dead chickens were dissected in time. The survival curve is shown in Table 7 and Figures 4-8 , wherein, Figure 4 is a survival curve; Figure 5 is a dissection graph of 1-day-old SPF chickens immunized with SCAU-D90 and challenged with virulent JL21 strain at 14 days old for 10 days; Figure 6 is a dissection graph of challenge control SPF chickens challenged with virulent JL21 strain at 14 days old and died 3 days later; Figure 7 is a dissection graph of 1-day-old SPF chickens immunized with SCAU-D90 and challenged with virulent FJ22 strain at 14 days old for 10 days; Figure 8 is a dissection graph of challenge control SPF chickens challenged with virulent JL22 strain at 14 days old and died 4 days later.

[0079] Table 7 Results of immune protection test of different epidemic strains

[0080]

[0081] From Table 7 and Figure 4 and Figure 5It can be seen that 1-day-old SPF chickens were immunized with SCAU-D90, and 14-day-old chickens were challenged with virulent JL21 strain. Ten days after challenge, the test chickens had no clinical symptoms, and the size and structure of the kidneys were normal. The vaccine protection rate was 100%. The 14-day-old SPF chickens in the challenge control group were challenged with virulent JL21 strain, and 3 days after challenge, the chickens showed signs of disease and death. The kidneys were enlarged and uric acid was deposited. The morbidity rate was 100%. The 1-day-old SPF chickens were immunized with SCAU-D90, and 14-day-old chickens were challenged with virulent FJ22 strain. Four days after challenge, one chicken died, and the other chickens showed no obvious symptoms. Ten days after challenge, all surviving test chickens had normal size and structure of the kidneys. The vaccine protection rate was 91.6%. The 14-day-old SPF chickens in the challenge control group were challenged with virulent FJ22 strain, and 4 days after challenge, the chickens showed signs of disease and death. The kidneys were enlarged and had flower spots. The morbidity rate was 100%. The results showed that the two challenge control groups had 100% morbidity and varying degrees of death after challenge, while the immunized groups had no obvious symptoms and pathological changes after challenge. The attenuated vaccine strain SCAU-D90 could effectively protect against two strains of GI-19 type, with a protection rate of more than 90%.

[0082] 7. Whole genome sequencing of attenuated vaccine strain SCAU-D90

[0083] SCAU-D90 has completed whole genome sequencing, and its whole genome sequence is shown as SEQ ID NO: 1. The nucleotide sequence encoding S protein is shown as SEQ ID NO: 2, and the amino acid sequence of s protein is shown as SEQ ID NO: 3. The nucleotide sequence encoding N protein is shown as SEQ ID NO: 4, and the amino acid sequence of N protein is shown as SEQ ID NO: 5.

[0084] The obtained strain was deposited, and the deposited name of the strain was chicken infectious bronchitis virus SCAU-D90 strain, the depositing time was April 28, 2024, the depositing number was CCTCC NO: V202450, and the depositing unit was China Center for Type Culture Collection, located in Wuhan, Wuhan University, China.

[0085] Example 3 Preparation of attenuated vaccine SCAU-D90

[0086] 1. Preparation of IBV virus liquid

[0087] The SCAU-D90 toxin obtained in Example 2 was diluted 1000 times with sterilized physiological saline, and 100 of the diluted solution was inoculated into the allantoic cavity of 10-day-old SPF chicken embryos, 0.2 mL per embryo. The embryos were sealed, incubated at 37°C, and observed once a day. The dead embryos were removed, and the remaining embryos were observed once every 4 hours. At 36 hours, all the embryos were removed, and the air sacs were placed upright at 4°C for 12 hours. The cooled embryos were removed, and the air sacs were sterilized. Then, the air sacs were removed from the shells by sterile surgery, the shell membranes were removed, the chorioallantoic membranes and amniotic membranes were cut, and the chorioallantoic fluid was collected for testing. The chorioallantoic fluid was stored at -20°C for later use.

[0088] 2. Test of IBV toxin The harvested virus liquid was tested for sterility and hemagglutination by the method described in the Chinese Veterinary Pharmacopoeia. A sample was taken for determination of virus content (EID50). The results showed that the virus liquid was sterile and negative in the hemagglutination test. The virus content was 10 7.0 EID 50 The above.

[0089] 3. Preparation and packaging of vaccine The virus liquid that passed the test was filtered through 4 layers of sterilized gauze, mixed in the same container, and mixed with an equal amount of 5% sucrose skim milk as a stabilizer. Penicillin was added at a dosage of 1000 units per mL, and the mixture was shaken well and packaged. The virus content was 10 4.0 EID 50 EID50 per 0.1 mL, and 1000 flocks per bottle. Then, the vaccine was freeze-dried, capped, and labeled, and stored at -15°C or below.

[0090] 4. Safety test of vaccine Ten 1-day-old SPF chickens were immunized with 10 times the immunization dose, and the safety of the vaccine was tested. The vaccine was diluted with sterilized physiological saline, and 200 μL (containing 10 flocks of vaccine) was dropped into the nostrils of each of the 10 SPF chickens. At the same time, 10 chickens were used as a control. The chickens were raised in a negative pressure isolator, and were allowed to drink and eat freely. The health of the chickens was observed and recorded daily, and the observation was continued for 21 days. The results showed that the chickens in the immunized group and the control group did not have any disease, and there was no difference in weight gain, indicating that the vaccine was safe.

[0091] 5. Efficacy test of vaccine

[0092] 30 1-day-old SPF chickens were divided into two groups. One group was immunized with 1 flock of the SCAU-D90 live vaccine prepared above, and the other group was not treated as a control. The chickens were raised in a negative pressure isolator, and were allowed to drink and eat freely. The health of the chickens was observed and recorded daily, and the chickens were challenged with the virulent SCAU-D5 virus 2 weeks later. Each chicken was challenged by dropping 10 4.0EID50 / 0.2mL, after challenge, the chickens were observed daily for morbidity and mortality, and the dead chickens were necropsied in time. After 2 weeks of observation, the surviving chickens were necropsied, and the pathological changes of organs such as kidney and trachea were recorded.

[0093] After immunization, the chickens in the experimental group and the control group were normal. After challenge, the chickens in the control group showed respiratory symptoms on the third day, some chickens were listless, and 4 chickens died. The surviving chickens had different degrees of tracheal bleeding, tracheal mucus, and kidney enlargement and pallor. The morbidity was 100% (15 / 15), and the mortality was 26.7% (4 / 15). The chickens in the immunized group were healthy and normal, indicating that the live vaccine prepared from the attenuated SCAU-D90 virus is safe and effective, and can protect against the challenge of homologous virulent virus.

[0094] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. GI-19 type chicken infectious bronchitis virus attenuated vaccine strain, characterized in that, The GI-19 type chicken infectious bronchitis virus attenuated vaccine strain is the chicken infectious bronchitis virus SCAU-D90 strain, which was deposited in the China Center for Type Culture Collection on April 28, 2024, with the deposit number: CCTCC NO: V202450, and the deposit address is: Wuhan University, Wuhan, China.

2. The GI-19 type chicken infectious bronchitis virus attenuated vaccine strain according to claim 1, characterized in that: The complete genome sequence of the SCAU-D90 strain of avian infectious bronchitis virus is shown in SEQ ID NO:

1.

3. The GI-19 type chicken infectious bronchitis virus attenuated vaccine strain according to claim 1, characterized in that The SCAU-D90 strain of chicken infectious bronchitis virus is obtained by attenuating the GI-19 type SCAU-D5 strain of chicken infectious bronchitis virus through 90 generations of chicken embryo passage.

4. The GI-19 type chicken infectious bronchitis virus attenuated vaccine strain according to claim 1, characterized in that: The nucleotide sequence of the S protein encoded by the SCAU-D90 strain of avian infectious bronchitis virus is shown in SEQ ID NO: 2, and the amino acid sequence of the S protein is shown in SEQ ID NO:

3.

5. The GI-19 type chicken infectious bronchitis virus attenuated vaccine strain according to claim 1, characterized in that: The nucleotide sequence of the N protein encoded by the SCAU-D90 strain of avian infectious bronchitis virus is shown in SEQ ID NO: 4, and the amino acid sequence of the N protein is shown in SEQ ID NO:

5.

6. A vaccine for preventing and treating GI-19 type chicken infectious bronchitis, characterized in that: The invention comprises the SCAU-D90 strain of chicken infectious bronchitis virus as claimed in any one of claims 1 to 5, and a pharmaceutically acceptable immunopotentiator and a freeze-dried protective agent.

7. Use of the SCAU-D90 strain of avian infectious bronchitis virus according to any one of claims 1 to 5 in the preparation of a medicament for preventing or treating avian infectious bronchitis.

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