CN106754746B - Avian infectious bronchitis virus vaccine strain, vaccine and application thereof - Google Patents

Abstract

The S strain can be directly applied to poultry as the attenuated vaccine to prevent the poultry from being infected and spread by the avian infectious bronchitis, the S strain has weak toxicity to SPF chickens, infected chickens do not have morbidity and death symptoms, are not easy to spread, have low toxin expelling rate and high safety, can carry out immune protection on domestic dominant IBV populations, have good effectiveness, and the SU strain is safe to SPF chickens, has the immune protection effect similar to the effects of L DT3 and H120 sold in the market, and has the possibility of being used as a safe vaccine candidate strain.

Description

Avian infectious bronchitis virus vaccine strain, vaccine and application thereof

Technical Field

The invention relates to the field of vaccines, in particular to an avian infectious bronchitis vaccine strain, a vaccine and application thereof.

Background

Avian Infectious bronchitis IB is a highly Infectious viral disease caused by avian Infectious Bronchitis Virus (IBV). Its highly pathogenic, contagious and globally widespread characteristics make it a virological respiratory disease that has been extensively studied in addition to newcastle disease and highly pathogenic avian influenza. Avian infectious bronchitis virus is infectious to chickens of different ages in days, and tropism to different tissues enables infected parts not to be limited to respiratory tracts, and parts such as stomach, intestinal tracts, kidney and fallopian tube can be affected (Cavanagh 2007). The pathogenic rate of the avian infectious bronchitis virus is up to 100%, and the death rate of infected chicks is 25% -30% (Bande, 2015). Infected chicks have an increased mortality rate if mixed or secondary infections occur with other bacterial or viral pathogens (Matthijs, 2003). Since 1931, it was first discovered that outbreaks of avian infectious bronchitis have been reported in many countries worldwide (Schalk, 1931). The disease frequently erupts in China after the 90 s in the 20 th century till now, so that the quality of broiler chickens infected with chicken flocks is reduced, the egg yield of laying hens is reduced, and great economic loss is brought to the poultry industry in China.

Avian infectious bronchitis viruses belong to the order Nidovirales (Nidovirales), the family Coronaviridae (Coronaviridae), the genus coronaviruses (Coronavirus). The virus is a single-stranded positive-strand RNA virus with an envelope, which is not segmented. The diameter of the virus particle is 80-120nm, and the surface of the cyst membrane is uniformly distributed with fiber protrusions with the length of about 20 nm. The major structural proteins encoded by the viral genome are spike (S), matrix (M), nucleocapsid (N) and envelope (E) proteins (Bande, 2015). The S protein is a highly glycosylated transmembrane protein with a size of 150-200 kDa. The protein plays a key role in host cell receptor recognition, antibody induction, pathogenicity, genotyping, etc. (Bande, 2015). The S protein gene is cleaved post-translationally into two subunits, S1(520aa) and S2(625 aa). Among them, the S1 protein recognizes and binds to host cell receptors, and the S2 protein fuses viral envelope and host cell membrane by conformational change, allowing viral genome to enter host cells (Belouzard, 2012). The S protein can induce the body to produce corresponding antibodies and immune response, while in the two glycoprotein subunits S1 and S2, S1 is the main immunogenic protein and has a plurality of antigen epitopes (Ignjatovic, 1991; Ignjatovic, 2005). The S1 protein likewise plays a crucial role in receptor binding and cell membrane fusion (Belouzard, 2012).

Traditional serological experiments such as virus neutralization experiments (VN) have found that avian infectious bronchitis viruses have multiple serotyping (OIE,2008) and their antigenic diversity has made it impossible to produce effective cross-protection between different strains of avian infectious bronchitis viruses (Farsang, 2002; L adman, 2006). therefore, for different serotypes of avian infectious bronchitis viruses, there are on the market various attenuated live vaccines and inactivated vaccines (Bijlenga, 2004). at present, attenuated live vaccines are widely used for the prevention and control of avian infectious bronchitis. live vaccines generally select strains of avian infectious bronchitis viruses with good antigenicity, such as MASS type M41 or H52, H120, etc., which are the first generation of products for use in the prevention and control of avian infectious bronchitis epidemic (L ee, 2010). inactivated vaccines are generally used in combination with attenuated live vaccines or alone.

It was found that genetic differences among individuals affect the efficacy of immune responses (Cavanagh,2007) and that 2-3% changes in the amino acid sequence of the S1 protein of avian infectious bronchitis virus (10-15aa) lead to changes in serotypes, thus generating variant strains and possibly affecting cross immune protection between similar serotypes (Cavanagh,1997) therefore, the S1 protein sequence of avian infectious bronchitis virus becomes the research focus for the prevention and control of avian infectious bronchitis, avian infectious bronchitis viruses distributed mainly around the world are also classified into classical strains and variant strains (Chang-Won L ee,2003) based on the sequence analysis of the S1 gene, wherein classical strains M41 and H120 are widely used as vaccine strains (Sjaak de Wit, however, the world animal health Organization (OIE) proposed that the use of avian infectious bronchitis virus serotypes have marginal influence on the use of avian infectious bronchitis in general in specific regions, such as strains M2, Arak 2 and C2011 (OIE), while the distribution of avian infectious bronchitis viruses is greatly challenging the spreading of infectious bronchitis viruses across geographical variations in European strains Sjaak, such as Sjaak, No. 2, Arabidopsis, 2011, No. 12, Shi.

The epidemiological investigation and research data of the avian infectious bronchitis virus in China discover that the prevention and control of the avian infectious bronchitis in China have the following characteristics: 1. the avian infectious bronchitis virus strains widely prevalent at present in China are mainly QX genotypes; 2. at present, Mass type vaccine strains such as H120, H52, Ma5 and M41 are generally used for preventing and controlling avian infectious bronchitis in China. But the Mass type has a far evolutionary relationship with the domestic popular genotype, so that the cross protection is poor; 3. there are relatively few commercial vaccines for the prevention and control of the QX avian infectious bronchitis virus.

Disclosure of Invention

In view of the above, there is a need to provide an avian infectious bronchitis virus vaccine strain, a vaccine and applications thereof.

An avian infectious bronchitis virus vaccine strain S with a preservation number of CCTCC NO: v201647. The vaccine strain S belongs to avian infectious bronchitis virus IBV. The preservation date of the vaccine strain S is 2016, 9 and 16 days, the preservation unit is totally called China center for type culture Collection, the preservation unit is abbreviated as CCTCC, and the preservation address is Wuhan city Wuchang Lojia mountain.

An avian infectious bronchitis vaccine composition comprises vaccine strain S and a pharmaceutically acceptable adjuvant or carrier.

The vaccine strain S is applied to preparation of the avian infectious bronchitis vaccine.

The vaccine strain S is applied to the preparation of protein or polypeptide used as immunogen.

The vaccine strain S is applied to the preparation of protein antigen for detecting specific antibody in serum.

An avian infectious bronchitis virus vaccine strain SU with a preservation number of CCTCC NO: v201648. The vaccine strain SU belongs to avian infectious bronchitis virus. The preservation date of the vaccine strain SU is 2016, 9 and 16 days, the preservation unit is totally called China center for type culture Collection, the preservation unit is abbreviated as CCTCC, and the preservation address is Wuhan city Wuchang Lojia mountain.

An avian infectious bronchitis vaccine composition comprises a vaccine strain SU and a pharmaceutically acceptable adjuvant or carrier.

The vaccine strain SU is applied to preparation of avian infectious bronchitis vaccines.

The vaccine strain SU can be used for preparing protein or polypeptide used as immunogen.

The vaccine strain SU can be used for preparing a protein antigen for detecting a specific antibody in serum.

The inventor selects partial pathological materials which are possible to generate the avian infectious bronchitis virus strain according to the origin place and the source year of the pathological materials, and carries out virus separation, animal regression experiments and S1 gene sequence comparison again to obtain a QX genotype avian infectious bronchitis virus natural vaccine strain which is named as S (S strain for short). The S strain can be directly applied to preventing poultry from being infected and spread by the avian infectious bronchitis virus as an avian infectious bronchitis virus vaccine. The S strain has weak toxicity to SPF chickens, does not cause morbidity and death symptoms when infecting the chickens, is difficult to spread, has low toxin expelling rate and high safety; the S strain belongs to CHI branch, namely QX genotype, can carry out immune protection on domestic dominant IBV population, and has good effectiveness.

In order to further obtain an avian infectious bronchitis virus vaccine strain with better effect, an extreme dilution method is used for quickly purifying an S strain and adapting a chick embryo to obtain the vaccine strain capable of preventing QX genotype avian infectious bronchitis, and the vaccine strain is named as SU (SU strain for short).

Drawings

FIG. 1 is a diagram showing the lesion after infection of chick embryos by S strain.

FIG. 2 is a diagram showing the result of RT-PCR identification of the N gene, wherein lane M is a DNA Marker D L1000, lane 1 is an amplification product of the N gene in the IBV portion, lane N is a negative control using water as a template, and lane P is a positive control using the H120 genome as a template.

FIG. 3 is a diagram showing the result of RT-PCR identification of M gene, wherein lane M is DNA Marker D L2000, lane 1 is the amplification product of the M gene in the IBV part, lane N is a negative control using water as a template, and lane P is a positive control using H120 genome as a template.

FIG. 4 is a particle morphology of S strain virus under an electron microscope.

FIG. 5 is a diagram showing the result of RT-PCR identification of the S1 gene, wherein lane M is DNA Marker D L2000, lane 1 is the result of RT-PCR amplification of the S1 gene, lane N is a negative control using water as a template, and lane P is a positive control using the H120 genome as a template.

FIG. 6 is a PCR result chart of the positive clone bacterial liquid, wherein lane M is DNA Marker D L2000, lanes 1-3 are PCR bands of the bacterial liquid, and lane N is a negative control using water as a template.

FIG. 7 is a diagram showing the results of genetic evolution analysis of S strain S1 gene and 34 reference strains.

FIG. 8 is a diagram showing the results of genetic evolution analysis of the entire genome sequence of S strain.

FIG. 9 is a graph showing the result of Western Blot detection of virus particles in allantoic fluid of SU strain. Wherein, Lane M is Marker, Lane N is chick embryo allantoic fluid inoculated with PBS, Lane P is allantoic fluid containing H120, and Lane 1 is a positive sample appearing during passage.

FIG. 10 is a diagram showing the morphology of SU strain virions under an electron microscope.

FIG. 11 shows deletion of SU strain compared with S strain.

Fig. 12 shows kidney pathology sections at day 5 after immunization of 3-day-old SPF chicks at 200-fold magnification, where fig. 12(a) is L DT3 immunization group, fig. 12(b) is SU immunization group, fig. 12(c) is H120 immunization group, and fig. 12(d) is non-immunization group.

Fig. 13 is the detoxification rate of the parent strain SF 5 days after challenge.

FIG. 14 shows the detoxification rate of heterologous strain M41 after 5 days of challenge.

Detailed Description

For a better understanding of the present invention, reference is made to the following detailed description taken in conjunction with the accompanying drawings. The reagents or instruments used in the present invention are commercially available, and the detection methods and the like used are well known in the art and will not be described herein.

Example 1 isolation and identification of S Strain

(1) Isolation culture of S Strain

Tracheal, bronchial, lung and kidney tissues from 2009 Hubei, 2009 Hunan, 2009 and 2010 Sichuan, 2009 Guangdong and 2010 Guangxi sick chicken were selected as disease samples. Inoculating the supernatant of the treated homogenized pathological material to SPF chick embryos of 9-11 days old through an allantoic cavity, continuously conducting blind propagation for 3 generations to obtain allantoic fluid containing IBV, inoculating the allantoic fluid to SPF chick embryos of 9-11 days old, incubating for about 48h at 37 ℃, extracting the allantoic fluid, and obtaining a separated strain.

(2) Identification of IBV

a) The strain is separated and inoculated with SPF chick embryos of 9-11 days old through an allantoic cavity path, and after the chick embryos are cultured for 6-7 days at 37 ℃, dwarfing embryos appear in the non-dead chick embryos, and the result is shown in figure 1.

b) Separating strain, inoculating SPF chick embryo of 9-11 days old via allantoic cavity as test group, inoculating SPF chick embryo of 9-11 days old via allantoic cavity with water as control group, incubating at 37 deg.C for 10-12h, and inoculating 100 × EID via allantoic cavity₅₀Newcastle Disease Virus (NDV)0.1 ml/embryo, incubation was continued for 48h for virus harvest and the hemagglutination titer (HA) of allantoic fluid was determined. Test ofThe hemagglutination price of the group chicken embryo (IBV + NDV) is 1-5log2, the hemagglutination price of the control group chicken embryo (NDV) is 8-9log2, interference exists, and avian infectious bronchitis virus is preliminarily determined to be contained in the disease material.

c) Extracting virus RNA in chick embryo allantoic fluid after blind passage of 3 generations, and performing RT-PCR to identify the virus by using primers designed aiming at conserved segments of M gene and N gene of avian infectious bronchitis virus genome. A negative control group using water as a template and a positive control group using an H120 genome as a template are simultaneously established. The specific sequence of the upstream primer NpF designed for the conserved segment of the N gene is CGGAGCAATAGCAAGAAAAGC, and the specific sequence of the downstream primer NpR is GCAGCAACCCACACTATACCATC. The specific sequence of the upstream primer MpF and the specific sequence of the downstream primer MpR, which are designed aiming at the M gene conserved segment, are CCTAAGAACGGTTGGAAT and TACTCTCTACACACACAC respectively.

The RT-PCR results are shown in FIGS. 2 and 3. As can be seen from FIGS. 2 and 3, the sizes of the bands of the N gene and the M gene of the test group (IBV) are about 500bp and 740bp, respectively, and are consistent with the size of the target fragment, and the isolated strain is further proved to be the avian infectious bronchitis virus.

(3) Virus particle morphology

The allantoic fluid extracted in step (1) was concentrated, and the morphology of virus particles was observed by electron microscopy (FIG. 4), which revealed that S-strain virus is mostly elliptical or spherical in shape, and has a diameter of about 100nm, and may be polymorphic. The virus surface fibers are uniformly arranged and show radioactivity.

(4) Evolutionary analysis based on avian infectious bronchitis virus S1 gene sequence

Primers are designed aiming at conserved sequences outside two ends of the S1 gene, the specific sequence of the upstream primer S1F is TTGAAAACTGAACAAAAGACCG, and the specific sequence of the downstream primer S1R is TACAAAACCTGCCATAACTAACAT.

PrimeScript by Takara^TMII 1st Strand cDNASynthesis Kit reverse transcriptase Specification for cDNA Synthesis. Preparing a reaction system by PCR amplification according to the instruction of KOD Plus Neo DNA polymerase, and pre-denaturing at 94 ℃ for 1 min; 30 cycles of 94 ℃ for 30s, 55 ℃ for 30s, and 68 ℃ for 54 s; extending for 5min at 68 ℃; and terminated at 4 ℃. The PCR amplified S1 gene product was detected by 1% agarose gel electrophoresis to yield about 1.7kbThe band of interest, the size of the band, was consistent with the positive control using the H120 genome as template (FIG. 5).

After the PCR product was recovered and purified by HiPure PCR Pure Micro Kit (magenta), TA cloning was performed according to the instruction of pMD19simple T Vector of Takara corporation, positive transformants (FIG. 6) were selected and sequenced, the nucleotide sequence of the S1 gene (without UTR) is shown in SEQ ID NO: 1, the whole gene sequence of S1 was submitted to NCBI B L AST SERVER for sequence homology search and identification, B L AST results showed that the S1 gene of the S strain has a 99% similarity to the strain isolated in China, which is very similar to CK/CH/SC/MS11-4 isolated in Sichuan and many different isolates isolated in Zhejiang, and the isolate from Anhui, Jiangsu, Guangxi, Guangdong was scored slightly lower.

34S 1 genes of isolated strains and common vaccine strains in China and all over the world are selected from a GenBank database to be used as reference sequences (Table 1) and are compared with the S1 gene of the S strain, the cluster and MEGA6.0 analysis software is used for phylogenetic analysis, an adjacent phase Joining method (Neighbor-Joining) is adopted to construct an evolutionary tree, and Bootstrap value is used for evaluating the reliability of the evolutionary tree (figure 7). The results show that 35 IBV S1 gene sequences are respectively clustered to form 8 clusters, the S strain and the domestic typical QX IBV are clustered to be CHI branches and clustered to be different branches with L DT3, and the domestic common vaccine strains H120, H52 and W93 are clustered to be Mass genes and clustered to be different large branches with the domestic isolate strains, and have far genetic relationship.

TABLE 1 Genbank 34 IBV reference sequences

(5) S strain whole genome cloning

Referring to the published IBV whole genome sequence in GenBank, classical representative strains in a plurality of serotypes are selected for sequence comparison analysis, and 10 pairs of primers for amplifying avian infectious bronchitis virus genes are designed by utilizing a section with high conservation degree in the IBV genome (Table 2).

TABLE 2 genomic PCR amplification primers

The PCR amplification parameters are 94 ℃ for 1min, 94 ℃ for 30S, 55-65 ℃ for 30S, 68 ℃ for 45S-1min, 30 cycles, 68 ℃ for 5min, 4 ℃ for termination, PCR product recovery purification and connection are generally 1kb/30S according to different fragment length extension time, the connection product is converted into a competent cell, and the positive colony identification and sequencing method are as described above, the full-length of the S strain obtained by sequencing is spliced and arranged by using STAR L asergene.v7.1 analysis software, and the whole gene sequence of the strain is submitted to NCBI B L AST SERVER for sequence homology retrieval and identification.

Reference strains for the comparison and analysis of the whole gene sequences of the S strains are all from GenBank (Table 3), multiple sequence alignments are respectively carried out on 1a, 1b, S1, S2, 3a, 3b, E, M, 5a, 5b and N11 genes by using Clustalx software, phylogenetic analysis of each gene is carried out by using MEGA6.0 analysis software, and reliability of the evolutionary tree is evaluated by using Bootstrap value after the evolutionary tree is constructed by using an adjacent phase Joining method (Neighbor-Joining) (FIG. 8). The gene sequences of 1a and 1b of the S strain are shown as SEQ ID NO: 3-4, and the S2, 3a, 3b, E, M, 5a, 5b and N gene sequences of the S strain are respectively shown as 5-12. The results show that the S strain has a very close relationship with the domestic isolates. The sources of the isolated strains are mostly distributed in Sichuan, Shandong and Heilongjiang. The original disease of S strain is from Sichuan, so the evolution of S strain has close relation with IBV strain circulating in Sichuan area.

TABLE 3 Whole genome analysis of representative strains from Genebank

Example 2 infection of half of S-strain chick Embryos (EID)₅₀) Pathogenic and safety tests

(1) Half of the infection amount (EID) of S strain chick embryo₅₀)

The allantoic fluid (virus fluid) extracted in step (1) of example 1 was diluted with a 10-fold gradient, and 6 SPF chick embryos were aseptically inoculated by allantoic cavity inoculation with an appropriate dilution gradient, and incubated at 37 ℃. Observing the chick embryos once every 12h, discarding the chick embryos dead in 24h, continuously observing for 144-168h, immediately refrigerating the dead chick embryos at 4 ℃, taking out all the chick embryos after the observation, recording the number of the dead embryos, the dwarfing embryos and the sinusoidal embryos as infection, recording the number of the infected embryos of each dilution, and calculating the EID50 of the virus to be 10 by using a Reed-Muench method^-5.5/ml。

(2) Pathogenic power of S plant

Taking S strains of SPF chickens of 5 days old as a test group by eye dropping and nose dropping for counteracting toxic substances, and taking 10^5.0EID₅₀Animals regression experiments using vaccine strains H120, L DT3 eye drops of nasal challenge as positive controls, 10^3.5EID₅₀And/or feather. The infection and mortality rates were calculated by daily observation. Collecting throat swab and anus swab 5 days after artificial infection to perform RT-PCR detection for detoxification. After 7 days, all live chickens were killed and examined for lesions in the trachea, lungs, and kidneys (Table 4). During the experiment, the test group broilers did not show any clinical symptoms or transient response. The chickens only eat the feed and are normal in spirit, which indicates that the S strain has no obvious pathogenicity on SPF (specific pathogen free) chickens of 5 days old. Compared with a control strain, the S strain has the toxin expelling rate of 50 percent and the morbidity mortality rate of 0, which indicates that the S strain can be the natural vaccine strain of the avian infectious bronchitis virus.

TABLE 4 results of the determination of the pathogenicity of different IBVs

(3) Safety of S strain

Dripping allantoic fluid of S strain for SPF chicken of 5 days age into eye, and dripping into nose for counteracting toxic substance at a dose of 10^5.0EID₅₀. After the challenge, the disease condition of the chickens is observed every day. After 5 days, the visceral lesion of the chicken is observed by killing, the kidney, trachea and lung grinding fluid is taken to be centrifuged for 3min at 8000rpm and 4 ℃, and the supernatant is taken to be subjected to RT-PCR detection. If the RT-PCR test is negative, the supernatant is addedInoculating to SPF chick embryo of 9-11 days old, incubating at 37 deg.C for 48h, aseptically collecting allantoic fluid, and performing RT-PCR detection. Rejuvenating with IBV-containing viscera homogenate supernatant or allantoic fluid obtained from chick embryo propagation, and repeating above operation for 10 times for 3-7 days SPF chicks by dropping eyes and nose.

20 SPF chickens of 3 weeks old were divided into 3 groups. One group was negative, and the chickens were subjected to PBS eye-drop nasal drip. Another group was challenged with S-strain passaged 5 and 10 times on chickens at a dose of 10^5.0EID₅₀. After the challenge, the state of the chickens is observed, only 1 of 10 chickens in a 5-time challenge group (abbreviated as S-5) appears with hoarse voice symptom after 3 days after the challenge, one cachexia symptom appears again on 4 days, and 1 dead chicken appears on 5 days, but no visual lesion appears after the cesarean examination of all the chickens. 6 infected chickens appeared in the challenge group (abbreviated as S-10) after 10 passages, white water samples were discharged, the sounds of the infected chickens were dull, 2 dead chickens appeared on the 5 th day, the kidneys of the dead chickens were found to be slightly swollen after the cesary examination, and the live chickens were not obviously changed. On the 5 th day after challenge, all chickens were swab-collected and viscus-collected, infection rates were detected, and infection and death conditions after challenge were recorded (table 5), indicating that the S strain returned strong virulence, but still within the safe range, and suitable for use as an avian infectious bronchitis virus vaccine.

TABLE 5 virulence test of S strains in different generations

Example 3 isolation and identification of SU Strain

(1) Limiting dilution passage method

Diluting allantoic fluid of S strain by 10 times gradient, and selecting and mixing with its EID₅₀Diluting with the same order of magnitude and 2 dilutions 10 times and 100 times higher than the dilution, inoculating 9-11 day-old SPF chick embryos, incubating at 37 ℃ for 48h, and aseptically collecting allantoic fluid. Centrifuging at 4 deg.C and 8000rpm for 15min, collecting supernatantWestern Blot detects avian infectious bronchitis virus (FIG. 9). The size of the target band is consistent with that of the band taking H120 as a positive control, and the sample is proved to contain the avian infectious bronchitis virus. The dilution of the positive allantoic fluid in the highest dilution containing only one or two positive bands was further diluted 10-fold and 100-fold next as the first dilution, and the above chick embryo inoculation and allantoic fluid collection and WesternBlot test procedures were repeated for 3-5 generations.

(2) Screening of candidate strains for SU vaccine

And (3) carrying out S1 sequencing comparison on the original strain, the 3 rd generation virus and the 5 th generation virus obtained after the extreme dilution passage and the common passage method passage. Limiting dilution passage method after sequencing of S1 gene of the strain obtained after the 3 rd and 5 th short passages, 2 point mutations causing amino acid changes were found in the sequence alignment of S1 gene of the primary S strain in 10 picked clones (Table 6). In the strains after 5 passages by using a common passage method, the sequence of S1 gene of 10 picked clones is aligned with that of a primary S strain, 8S 1 genes are identical with that of a parent strain S, and T → C point mutation exists at the position 81 in the other 2 clones, but the mutation does not bring amino acid mutation. The 5 th strain of the extreme dilution passage method is selected and named as SU and is proposed as a vaccine candidate strain.

TABLE 6 comparison of the S1 Gene sequences of SU and S strains

(3) Virus particle morphology

IBV allantoic fluid was concentrated, and the morphology of virus particles was observed by electron microscopy (FIG. 10), whereby it was found that SU strain virus is mostly elliptical or spherical in shape, has a diameter of about 100nm, and may be polymorphic. The virus surface fibers are uniformly arranged and show radioactivity.

(4) Whole genome sequencing analysis of SU Strain

The methods of extraction of the genome of the SU strain virus, synthesis of genomic cDNA, primer design of the S1 gene, PCR amplification of the S1 gene, cloning of the S1 gene, and sequencing were as described above.

The SU strains were subjected to whole genome sequencing analysis and their sequences were aligned with the parental strain S strain (table 7, fig. 11). Compared with the parent strain S strain, the sequence comparison shows that more point mutations appear in the 1a and 1b genes, the other more important structural proteins such as S1 and S2 have less mutations, the nucleotide mutation of the M gene does not introduce amino acid substitution, and the E, N gene does not have any point mutation. In Table 7, double-underlined marks indicate that the substituted amino acid remains a polar amino acid, wavy marks indicate that the substituted amino acid is substituted for a polar amino acid from a nonpolar amino acid, and dotted marks indicate that the substituted polar amino acid is changed to a nonpolar amino acid. The number of amino acid substitutions by SU due to nucleotide changes was 13, whereas the total of 7 substitutions with amino acid polarity changes, the nonpolar substitution was 2/7, the polar substitution was 5/7, and the probability of substitution was higher for the nonpolar amino acid. The sequence of the S1 gene of the SU strain is shown in SEQ ID NO: 2, respectively. Both point mutations of the S1 gene of SU strain resulted in a change of polar amino acid to nonpolar amino acid, which probably caused a change in the secondary structure of the S1 protein. In addition, complete comparison of the S-strain and SU-strain genomes revealed that the SU-strain had a deletion of 110bp in the 3' UTR after the termination codon of the N gene (FIG. 11).

TABLE 7 comparison of the complete genome sequences of SU and S strains

Example 4 chick embryo infection amount (EID) of SU Strain₅₀) Optimal immunization dose, safety experiment and effectiveness experiment

(1) Half of the infection amount (EID) of SU chick embryos₅₀) Measurement of (2)

The EID described in example 2 was used₅₀Measurement method, EID of SU Strain₅₀Measurement of (2), EID thereof₅₀Is 10^-7.37。

(2) Determination of optimum immunization dose of SU strain

Immunizing SPF chicken of 3 days old by eye dropping and nose dropping, collecting blood 21 days after immunization, detecting antibody level with E L ISA in serum, evaluating stimulation of SU strain on chicken immune system by antibody level in serum, and determining optimal immunization dose as 10^5.5EID₅₀。

(3) Safety test of SU Strain

Collecting SPF chicken of 3 days old, and 1-3 groups are SU immunization groups with immunization dose of 10^3.5EID₅₀、10^4.5EID₅₀、10^5.5EID₅₀Setting commercial vaccine strain L DT3 and H120 positive control group, and the immune dose is a recommended dose (10) of one feather^3.5EID₅₀) All SU immunization groups and H120 and L DT3 immunization groups do not have any IB clinical symptoms, and the states of the spirit, the food intake and the excrement are not different from those of the negative control group, a swab is taken after 5 days of immunization to carry out RT-PCR detection on the detoxification rate (Table 8), the result shows that the detoxification rate of the SU strains is low, and the SU strains are proved to have weaker toxicity to SPF chickens than L DT3 and H120 until 10 days of use^5.5The immune dose of EID50 only allows chickens to achieve near 100% detoxification rate.

TABLE 8, 3-day-old toxin expelling rate after SPF challenge

The section observation shows that the trachea, the lung and the kidney of the SU group and the H120 vaccine immunization group have no obvious pathological change compared with a negative control group which does not carry out immunization operation, but the section form of the kidney of the L DT3 immunization group is different from that of the non-immunized group, the renal tubule structure is loose, the renal tubule type is obvious, and the kidney may have certain damage.

Taking 1 day-old SPF chickens, and the immune doses of SU group are respectively 10^4.5EID₅₀、10^5.5EID₅₀、10^6.5EID₅₀Feather, chicken were observed daily (table 9). On day 3, it was observed that all chickens in each group had watery feces except for the control group, but the chickens were in good mental status and had no symptoms. On day 5, the swabs were collected, the chickens were voiced and clear without rale, snore, mouth-opening breathing, head-swinging, sneezing, etc., until day 21 no dead chickens appeared.

(4) Test of the effectiveness of SU Strain

Taking SPF chicken of 3 days old, and applying SU strain via eye-drop and nose-drop route to obtain a dosage of 10^5.5EID₅₀Immunization of the feather. 21 days after immunization, all groups used the corresponding challenge strain (parental strain SF or heterologous strain M41) at 10^5.0EID₅₀The virus attacking dose of feather is instilled into eyes and the nose to attack the virus, the state of the chicken is observed every day, and a throat swab and an anus swab are taken on the 5 th day after the virus attacking for RT-PCR detection to detect the toxin expelling rate, L DT3 and an H120 positive control group and a negative control group are simultaneously arranged, wherein, a parent strain SF is a virulent strain obtained by transmitting an S strain to the 14 th zone by a conventional passage method.

Challenge protection against parental strain SF (figure 13). During the observation period, the negative control group showed suspected clinical symptoms of mouth breathing and listlessness, and other chickens showed no obvious symptoms. The toxin expelling rate of the swab after 5 days of toxin attacking shows that SPF chickens of 24 days old are in SF strain 10^5.0EID₅₀After 5 days of feather attacking, the toxin expelling rate of the throat swab and the anus swab of the negative control group reaches 100 percent; the toxin expelling rate of the throat swab in the SU immune group is 81.25 percent, the toxin expelling rate of the anal swab is 35 percent, and the SU immune group has a better immune protection effect on SF strains.

Cross protection (figure 14) for the heterologous strain M41, observation period, negative control group appeared flail and listless suspected IB clinical symptoms, and the other chickens did not show obvious symptoms, after 5 days of toxin challenge, the toxin expelling rate of the SU immune group in the pharynx swab is 40%, the protection effect for the heterologous strain M41 is better than that of the L DT3 group, but still worse than that of the H120 group.

Taking another SPF chicken of 1 day old, and carrying out dosage by using SU strain through eye-dropping and nose-dropping ways10^4.5EID₅₀Feather, 10^5.5EID₅₀Feather sum 10^6.5EID₅₀Immunization of the feather. On day 3, it was observed that all chickens in each group had watery feces except for the control group, but the chickens were in good mental status and had no symptoms. On day 5, the swabs were collected, the chickens were voiced and clear without rale, snore, mouth-opening breathing, head-swinging, sneezing, etc., until day 21 no dead chickens appeared. 21 days after immunization, all groups used the corresponding challenge strain SF at 10^5.0EID₅₀The drug counteracting dose of each feather is instilled to the nose to counteract the drug, the state of the chicken is observed every day, and the death rate is counted (table 9). An H120 positive control group and a negative control group were also set.

TABLE 9, 1 day-old SPF chickens post challenge observation

The invention separates and identifies an avian infectious bronchitis virus strain S and SU to carry out genetic evolution analysis on the S1 gene of the S strain, and proves that the S strain belongs to QX type avian infectious bronchitis virus and can carry out immune protection on domestic dominant IBV populations. The full-length genome of the S strain except UTR at two ends is compared and analyzed with 25 avian infectious bronchitis virus reference viruses on GenBank, the evolution of the S strain is greatly influenced by domestic isolates, the S strain has far genetic relationship with Mass type vaccine strains and foreign isolates, and the S strain is probably a recombinant strain. The S strain can be directly applied to preventing poultry from being infected and spread by avian infectious bronchitis as a natural virus vaccine, has weak toxicity to SPF chickens, does not cause morbidity and death symptoms of infected chickens, is not easy to spread and has low toxin expelling rate.

The SU strain is subjected to safety tests and immunity efficacy tests, and the SU strain is proved to be safe to SPF chickens, has similar immune protection effect to that of L DT3 and H120 sold in the market and has the possibility of serving as a candidate strain of a safe vaccine.

The vaccine can be prepared by taking the S strain or the SU strain as a production virus seed, inoculating SPF (specific pathogen free) chick embryos of 9-11 days old, inoculating 0.2m L into each allantoic cavity, continuously incubating at 37 ℃, irradiating embryos for 48 hours after inoculation, discarding dead embryos, cooling the live embryos at 2-8 ℃, harvesting allantoic fluid containing the virus chick embryos, mixing the allantoic fluid with a common freeze-drying protective agent, packaging, and performing freeze vacuum drying to prepare the live vaccine for the infectious bronchitis chicken.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

<110> Zhongshan university

<120> avian infectious bronchitis attenuated vaccine strain, vaccine and application thereof

<160>12

<170>PatentIn version 3.5

<210>1

<211>1620

<212>DNA

<213> avian infectious bronchitis Virus (Infectious Bronchitis Virus)

<400>1

atgttgggga agtcactgtt tttagtgacc attttgtgtg cactatgtag tgcaaatttg 60

tttgattctg ccaataatta tgtgtactac taccaaagtg cctttaggcc tccaaatgga 120

tggcatctgc aagggggtgc ttatgcagta gtgaattcta ctaattatac taataatgcc 180

ggttctgcaa gtgggtgcac tgttggtgtt attaaggacg tctataatca aagtgcggct 240

tccatagcta tgacagcacc tcctcagggt atggcttggt ctaagtcaca attttgtagt 300

gcacactgta acttttctga aattacagtt tttgtcacac attgttatag tagtggtgca 360

ggttcttgtc ctataacagg catgattgca cgtgatcata ttcgtatttc tgcaatgaaa 420

aatggttctt tattttataa tttaacagtt agcgtatcta aataccctac gtttaaatct 480

tttcaatgtg ttaacaattc cacatctgtt tatctaaatg gtgatcttgt ttttacttct 540

aacaaaactg ctgatgttac gtcagcaggt gtgtattata aagcaggtgg acccgtaaat 600

tatagtgtta tgaaagaatt taaggttctt gcttactttg ttaatggtac agcacaagat 660

gtaattttgt gcgacaattc ccccaagggt ttgctggctt gtcaatataa tactggcaat 720

ttttcagatg gcttttatcc ttttactaat agtactttag ttagggacaa gttcattgtc 780

tatcgtgaaa gtagtgtcaa tactactttg acgttaacta atttcacttt tactaatgta 840

agtactgcac agcctaatag tggtggtgtt agtacttttc atttatatca aacacaaaca 900

gctcagagtg gttattataa ttttaatttg tcatttctga gtcagtttgt gtataaggca 960

agtgatttta tgtatgggtc ttaccaccct aggtgttctt ttagaccaga aaccattaat 1020

agtggtttat ggtttaattc cttgtcagtt tctcttactt atggacccct acagggaggg 1080

tgtaagcaat ctgtttttaa tggtaaggca acgtgttgtt atgcctactc ttataatggc 1140

ccaagggcat gtaaaggtgt ttattcaggt gaattaagca agacttttga atgtggattg 1200

ctggtttatg ttactaagag tgatggctct cgtatacaga ctagaacgga gcccttagta 1260

ttaatgcaac acaattataa taatattact ttagataagt gtgttgacta taatatatat 1320

ggcagagtag gccaaggttt tattactaat gtgactgatt ctgctgctaa ttttagttat 1380

ttagcagatg gtgggttagc tattttagat acttcgggtg ccatagatgt ctttgttgta 1440

cagggcagct atggtcttaa ttattacaag gtcaatcctt gtgaagatgt taaccaacag 1500

tttgtagtgt ctggtggcaa tatagttggc attcttactt ctagaaatga aacaggttct 1560

gaacaggttg agaaccagtt ttatgttaag ttaaccaata gctcacatcg tcgcaggcgt 1620

<210>2

<211>1620

<212>DNA

<213> avian infectious bronchitis Virus (Infectious Bronchitis Virus)

<400>2

atgttgggga agtcactgtt tttagtgacc attttgtgtg cactatgtag tgcaaatttg 60

tttgattctg ccaataatta tgtgtactac taccaaagtg cctttaggcc tccaaatgga 120

tggcatctgc aagggggtgc ttatgcagta gtgaattcta ctaattatac taataatgcc 180

ggttctgcaa gtgggtgcac tgttggtgtt attaaggacg tctataatca aagtgcggct 240

tccatagcta tgacagcacc tcctcagggt atggcttggt ctaagtcaca attttgtagt 300

gcacactgta acttttctga aattacagtt tttgtcacac attgttatag tagtggtgca 360

ggttcttgtc ctataacagg catgattgca cgtgatcata ttcgtatttc tgcaatgaaa 420

aatggttctt tattttataa tttaacagtt agcgtatcta aataccctac gtttaaatct 480

tttcaatgtg ttaacaattc cacatctgtt tatctaaatg gtgatcttgt ttttacttct 540

aacaaaactg ctgatgttac gtcagcaggt gtgtatttta aagcaggtgg acccgtaaat 600

tatagtgtta tgaaagaatt taaggttctt gcttactttg ttaatggtac agcacaagat 660

gtaattttgt gcgacaattc ccccaagggt ttgctggctt gtcaatataa tactggcaat 720

ttttcagatg gcttttatcc ttttactaat agtattttag ttagggacaa gttcattgtc 780

tatcgtgaaa gtagtgtcaa tactactttg acgttaacta atttcacttt tactaatgta 840

agtactgcac agcctaatag tggtggtgtt agtacttttc atttatatca aacacaaaca 900

gctcagagtg gttattataa ttttaatttg tcatttctga gtcagtttgt gtataaggca 960

agtgatttta tgtatgggtc ttaccaccct aggtgttctt ttagaccaga aaccattaat 1020

agtggtttat ggtttaattc cttgtcagtt tctcttactt atggacccct acagggaggg 1080

tgtaagcaat ctgtttttaa tggtaaggca acgtgttgtt atgcctactc ttataatggc 1140

ccaagggcat gtaaaggtgt ttattcaggt gaattaagca agacttttga atgtggattg 1200

ctggtttatg ttactaagag tgatggctct cgtatacaga ctagaacgga gcccttagta 1260

ttaatgcaac acaattataa taatattact ttagataagt gtgttgacta taatatatat 1320

ggcagagtag gccaaggttt tattactaat gtgactgatt ctgctgctaa ttttagttat 1380

ttagcagatg gtgggttagc tattttagat acttcgggtg ccatagatgt ctttgttgta 1440

cagggcagct atggtcttaa ttattacaag gtcaatcctt gtgaagatgt taaccaacag 1500

tttgtagtgt ctggtggcaa tatagttggc attcttactt ctagaaatga aacaggttct 1560

gaacaggttg agaaccagtt ttatgttaag ttaaccaata gctcacatcg tcgcaggcgt 1620

<210>3

<211>11862

<212>DNA

<213> avian infectious bronchitis Virus (Infectious Bronchitis Virus)

<400>3

atggcttcaa gcctaaaaca gggagcatct tcccaaaacc agaggagtgt cgttcttgtt 60

accaaagaca ttcccgacca actctgtgac gctttgtttt tctatacgtc acacaatcca 120

aaagattacg ctgatgcttt cgcctttagg cagaagttcg accgtagtct cggtgctggg 180

aaacagttta aatttgaaac tgtttgtggt ccattcttct tgaagggagt ggataaaata 240

acaccaggag tcccagccaa agttataaaa gccacttcaa agttggctga cttagaagac 300

atctttggtg tctcacctct tgcacggaaa taccgtgaac ttttgaaaac agcatgccat 360

tggtctctca ctgtagaagc gctggatgct cgtgcacaaa ctcttgaaga aatctttgaa 420

ccaactgaaa tattttggct tcaagtggct gcaaaggttc aggtttcagc catggcaatg 480

cgcagacttg ttggtgaggt tactgcaaaa gtcatggatt cacttggctc aaatttgagc 540

gctctgttcc aagttgttaa ggaaacaatg gccagaatct ttcaaaaggc tctggccatt 600

tttgagagtg tgagtgaatt gccacaacgt attgctgcac ttaagatggc ttttgccaag 660

tgtgcaagat taattacagt tgtggttgtt ggacatgccc tcactattag agagttttct 720

ggaacttgtc ttgcaagcat taatggtgca gttgcaaaat tctttgaaga acttccaact 780

ggctttatgg gcgctaaggt cttttcaact cttgccttct ttaaagaagc agcagtaaag 840

attgtagaaa atataccaaa tgcgccaaga ggtactaaag gttttgaagt cgttggcaat 900

gccaaaggta cacaagttgt tgtccgtggc atgcgaaacg acttaacttt gcttgaccaa 960

aaagctgaag ttcctgtgga atcagaaggt tggtctgcaa tcttggaggg acatctttgc 1020

tatgtcttta agagtggtga tcgcttttat gcggcacctc tttcaggaaa ttttgcattg 1080

catgatgtgc attgctgcga gcgtgtagtc tgtctgtctg atggtgtaac accggagata 1140

aatgatgggc ttgttcttgc tgcaatttac tcatctttta gtgtttcgga acttgtggca 1200

gcacttaaaa aaggtgaacc attcaagttc ttggggcaca aatttgttta tgcgaaggaa 1260

gcggcagttt ctttcacctt ggcaaaagca gcaaccattg gagatgtcct gaagctgttt 1320

caatcagcaa gagtaaagac gaaggatgtg tggtctgcac ttactgaaca gtcttttgaa 1380

ttctggaaac ttgcgtacaa tagagtgcgc aatcttgaag aagttctgaa gactcatttt 1440

tgtaaagctc aaatgtcagt tgtgatccta gcagtagtgc ttggtgaggg catttggcat 1500

ctctgttcac aagtcattta taaattgagt ggcttgtttt ccaaagttgt tgacttttgt 1560

gaaaaacact ggaaaggttt ctgcacacag ctgcgaaaag ctaggcttgt tgttacagaa 1620

aatctctgtg tgttgaaagg aattgctcag cattgttttc aacttttgct ggaagcaata 1680

cattccatgt acaagagttt taagaaatgt gcacttggta ggatttccgg tgatctcttc 1740

ttctggaaag gaggtgtgca taaaataatt cacgatggcg atgaaatttg gtttgacgcc 1800

gtcgatacta ttgaagttga agatttgggt accgtccaag aaaaacttat agattttgag 1860

gtttgcgagg atgtgatact tccagaaaat caacctggcc acatggttca aatccaggat 1920

gatggaaaga actatatgtt cttccgtttc aagaaggatg agaacattta ttatacacca 1980

atgtcacaac ttggtgctat taatgttatt tgcaaagcgg gtggcaagac tgtcacgttt 2040

ggtgaaacaa ctgtaaagga aataccacca cctgatgttg tgcctattag ggtaagcatt 2100

gagtgttgtg gtgaaccatg gaatacaatc ttcaagaagg cttataaaga gcccattgaa 2160

gttgagacag acctcacagt agaacaactg ctcactgtga tctatgataa aatgtgtgaa 2220

gatctcaaac tgtttccaga ggcaccagaa cctccaccat ttgagaatgt tgcacttgtt 2280

gataaaaacg gtaaagatct ggattgcata aaatcatgcc atctcatcta ccgtgattat 2340

gagagcgatg atgacatcga ggaagaagat gctgaggagt gtgatacaga tccagctgat 2400

gctgaggaat gtgatactgc ttcagagtgt gaagaagaag atgaagatac aaaggtgctg 2460

tctcttttgc aagacccagc cagtaacaag tatcctctac ctcttgatga tgattatagt 2520

gtctataatg gatgtattgt tcataaggac gctcttgatg ttgttaactt accatctggt 2580

gaagaaactt ttgttgtcaa taactgtttt gaaggggctg taaaaccgct tccgcagaag 2640

gttgttgatg tccttggaga ttggggagag gccgttgatg cacaagaaca attgtgtgaa 2700

caagagtctg aatctgttaa agaatcagtt gagaaatcta ctggtgttgg tggttcagca 2760

attgaagaag ctgttgttgt agagcaggaa gtagttcctg ttgttgaaga gtctcaagaa 2820

gtagttgttt ttacgcctgc tgatttagaa gttgctaaag aaacatcaga ggatgtcgac 2880

gagtttgttc ttgttgctga tgttcttaca gaaaaagttg tgcttcaaga aaaagaagag 2940

ccacaagtag aacaggagcc tattcaagtt gtcaaaccac aacgtgagaa aaaggctaaa 3000

aagtttaaag tcaggcaaac cacatgtgaa aaacccaaat tcttggagta cacaacatgt 3060

gtaggtgatt tgactgttgt aattgccaaa gcattggatg aatttaaaga gttctgcgtt 3120

gttaacgccg caaatgagca tatgtctcat ggtggtggcg ttgcgaaggc aattgctgac 3180

ttttgtggac ctgattttgt ggaatattgt gaggactatg ttaagaaaca tgggccacaa 3240

caaagacttg tcacaccttc gtatgtcaaa ggcattcaat gtgtgaataa tgttgtagga 3300

ccccgccatg gcgacaaaga tttggaagaa aaacttgtcg ctgcctacaa gagtgttctt 3360

gtagatggtg tagtcaatta tgttgtgcca gttctctcat cgggaatttt tggtgtagat 3420

tttaaaactt caatagacgc gatgcgtaag gcttttgaag ggcttaacat acgcgtgctt 3480

ctcttttctc tgagtcaaga acacatcgat tatttcgacg caacttgtaa gcagaagaaa 3540

atttatctta cagaagatgg tgttaaatac cgctctgttg tagtgaaacc tggcgactct 3600

ttgggtcaat ttggtcaggt ttttgcaaag aacaagacag tttttacagc agatgatgtt 3660

gaggacgaag aagttctctt cactccaaca actgacaaag ctgttcttga atactatgga 3720

ttggatgcgc aaaagtatgt aatatatttg caaacactcg cgcagaaatg ggaagtccaa 3780

tatagggaca attttatctt acttaaatgg cgtgatggaa actgctggat tagttcagct 3840

atggtgctcc ttcaggccgc taagattagg tttagaggtt ttcttgcaga ggcatgggct 3900

aaattcttgg gtggagacgc tacggatttt gttgcttggt gctatgcaag ttgcaatgct 3960

aaagttggtg atttttcaga ctgcaattgg cttttggcta acttggcaga acactttgat 4020

gcagattaca caaatgcact gcttaagaag cgtgtgtcgt gtaactgtgg tgttaagagt 4080

tatgaactta gaggccttga agcttgtatt caaccagtaa gggctcctaa ccttctacat 4140

tttaagacgc aatattcaaa ttgtccaact tgtggtgcaa atagtatgga tgaagttgtt 4200

gaagcatcac tcccgtactt gttgcttgtt gctactgatg gtcctgctgc agttgattgt 4260

gatgaaaatg ctgcaggaaa tgttgttttt attgggtcca ccaacagtgg acactgttat 4320

acacaagctg ttggaaaagc ttttgataat ttggctcaag ataggaaatt tggaaagaga 4380

tcgccttaca ttactgcaat gtatgtgcgc ttctctctta agagtcaaaa ttctctgtct 4440

gttgctaaaa agagtaagag taagagtgaa gtggtaaagg aagatgtttc taaccttgcc 4500

actagttcaa aattcagttt tgatgatctt actgactttg aacagtggta tgatagtaac 4560

atctatgaaa gtcttaaagt gcaggaaaca cctgagagta tgggtgagta tgtgtcattt 4620

acaacaaagg aagattccaa gttgccactg acacttaaag ttagaggtat caaatcaggt 4680

gttgacttta agtcaaagga tggctttact tataagttga cacctgacac tgacgaaaat 4740

tcaaatgcac ctgtctacta tcctactttg gactctgtaa gtcttaaagc aatatgggtg 4800

gacggtactg ctaattttgt tgttggacac cccaactata atagtagagc tctacgtatc 4860

cccacattct gggaatgcgc agagaccttt gtaaaaatag gcgaaaaagt tgatggtgta 4920

actatgggcc tttggcgtgc agaacacctt aatagaccta atttggaaag gattttcaac 4980

gtcgttaaga agacgatggt tggaactagt gttgttacta cacaatgtgg taaattaatt 5040

agtaaagcag ctacattcgt tgccgataaa gtaggagatg gtgtagttcg taatgttact 5100

gatagaatta aagggtgttt tgggtttact agggattact ttgaaagaag agtgtctaca 5160

cagttcctta aaaccctctt tttcttcttt ttctgtctct tgaaggctag tgttaagagt 5220

ttaatttcta gctacaagag tgtgttatgt aaagtggtat ttactgcttt atttatacta 5280

tggtttgtct acacaagcaa cccagttata tttactggaa tacgtgtgtc agacttttta 5340

tttgaaggtt cattctgtag tccttatgta gactacggta aagagtcttt tgatatttta 5400

cgctattgtg gaagtgattt cacttgtcgt gtctgtttac atggtaagga ttcgctccat 5460

ttgtataaac atgcctatag cgtagaacag ttttataaag atgcagttaa tggcattagt 5520

tttacatgga attggcttta tatgctgttc ctactattgt ttgttaagcc agtagcaggt 5580

tttgtcatta tttgctactg tataagatat ttggttttga gcacaactgt tttacaaact 5640

ggtgtaggat ttttagactg gtttatccaa acagtgtttg ctaactttaa ttttatgggt 5700

gcaggatttt atttctggct tttttataag gtgtatgtac aggtgcatca tattatgtac 5760

tgtaaggata taacatgtga agtgtgtaag cgagttgcac gcagtaatagacatgaggtt 5820

agtgttgttg ttaacgggcg taaacagctt gttcacgtct acactaactc aggctataat 5880

ttctgtaaga gacataattg gtactgtaga aattgtgata agtatggcca ccaaaataca 5940

tttatgtctc ctgaagttgc tggcgagctt tctgaaaagc ttaagcgcca tgttaaacct 6000

acagcattcg cataccacgt agtggatgat gcatgcttag ttgatgactt tgttaatttg 6060

aagtataatg ttgccattcc aggtaaagaa ggtgtgcatt ctgctgttaa atgttttagt 6120

gtatctgact tcttaaagaa agctgtcttt cttaaggatg cacaaaaatg tgagcaaata 6180

tctaatgata gttttatagt ttgtaataca cacagtgcgc atgcattgga ggaagcaaaa 6240

aatgctgcca tctattatgc gcaatgtctt tgtaaaccaa ttcttatact tgatcaggcc 6300

ctttacgaac aattggttgt agaacctgtg tcaaaaagtg tagtagataa ggtgtgtagc 6360

atcctgtcta atataatatc tgtagactct gcagctttga attataaggc tggcacactt 6420

cgtgatgccc tcctttctgt tactaatgat gaagaagccg tcgatatggc tatcttctgt 6480

cataataatg atgttgaata tactagtgat ggttttacta atgttgtgcc gtcatatggt 6540

atagatactg ataaattaac acctcgtgat agaggctttt tgataaatgc agatgctgcc 6600

attgctaact tgaaagtgaa aaattcaccg ccagtagtgt ggaagtactc tgatcttgtt 6660

aaactgtctg acagttgtct taagtattta atatcagcaa ctgtcaaatt gggaggtagg 6720

tttttcataa caaggtctgg tgctaaacaa gttattgctt gttatactca gaagttgtta 6780

gtggaaaaga aggctggtgg tgttgttagt agcactgtta gttggtttaa gagttgttgt 6840

aaatggctct tagtgtttta tttacttttt acagtgtgtt gtttgggtta ttatcaatgg 6900

gaaatgagta gagaatttgc tcctcccatg tatgatttta actccacaat gcatgtagaa 6960

ggatttaagg ttattgacaa gggtgtactc agggatatta tgccagaaga cacttgcttc 7020

tctaataagt atgttaattt tgacccattc tggggtaaac cttatgtaaa tagtagagac 7080

tgtcctattg taaccgctat tatagatggt tctggtacgg tggctgctgg tgttcctggt 7140

tttgtacagt gggtcatgga tggtgtaatg tttattcata tgacacagac tgagcgaaga 7200

ccgtggtata ttccgacttg gtttaataga gaaattgttg gttatactca tgattctatt 7260

attacagaag gtgagtttta tacatccata gcattgtttg ctgctagatg tttgtattta 7320

acatctagta atacaccaca actttattgt tttaatggtg ataatgatgc tcctggtgct 7380

ttgtcatttg caagtatact tccacataga gcatactttc aacctaatgg agttaggctt 7440

attgtgcctc aacaaattat gcacacacca tacatagtta agttagtttc agacagctat 7500

tgtagaggta gtgtttgtga ggttactaaa ccaggttatt gtgtctcaat gaactctcaa 7560

tgggttttgt ttaatgatga atacacaatt aaaccaggcg tgttttgtgg ttctactgtt 7620

agagaacttt tgtttaatat ggttagtaca ttctttacgg gtgttaatcc aaacatttat 7680

atgcaattgg caattatgtt tttaatactt gttgctgttg tgttagtctt tgcaatggtt 7740

ataaagtttc aaggtgtttt taaagcttat gcaaccactg tatttactat aatgcttgtt 7800

tgggttatta atgcatttgt attatgtgtg catagttata atagtgttct agctattata 7860

ctacttgtac tctattgtta tgcatcactt gtgacgagtc gtaacactgc tataataatg 7920

cactgttggc tcgtttttac ttttggttta atagtaccta tgtggatagc ttgttgttat 7980

cttgcgtttg tactttacat gtatacacca ctatttttct ggtgttatgg tactactaaa 8040

aattctcgta agctttatga tggcaacgag tttgttggta attatgatct tgccgcgaag 8100

agtacttttg tgattcgtgg tcccgaattt gtaaagctta cgaatgaaat aggtgataaa 8160

tttgaagttt acctctcagc gtatgctaga cttaaatact attcaggtac aggcagcgag 8220

caagattatt tgcaagcctg ccgtgcgtgg ttggcttatg cactagacca atttagaagt 8280

agtggtgtag aaatggttta tactccaccg cggtattcta ttggtgttag tagattacaa 8340

gctggtttta aaaaactagt tgctcctagt agtgctgttg agaagtgcat tgttagtgtc 8400

tcttatagag gcaataatct taatggattg tggttgggtg attccatcta ctgtccacga 8460

catgtattag gtaagtttag tggtgaccaa tggagtgatg tacttagcct tgctaataat 8520

tatgaatttg aggttgtaac tcataatggt gttactttga atgttgtcag caggcgttta 8580

aagggtgcag tactgatttt acagactgca gtagccaatg ctgatacacc gaagtataaa 8640

tttttgaaag caaattgtgg tgatagtttc acaatagctt gctcttatgg tggtacagtt 8700

ataggactct accccgttac tatgcgttct aatggaacta ttagagcatc gtttcttgct 8760

ggagcatgtg gttcagtagg ttttaatata gaaaagggtg tagtaaattt ttactacatg 8820

caccatctag agttacctaa tgcattacac acaggaactg acctaatggg tgagttttat 8880

ggtggttata tagatgaaga ggttgctcag aaagttcaac ccgataaatt agttactaat 8940

aatattttgg catggcttta tgcagcaatt attagtgtta aagagagtag tttttcaaca 9000

ccaaaatggc ttgaaagtac tactatcagt attgatgatt ataataagtg ggcaggtgat 9060

aatggtttta catcatttgt aagctgcact gctattacta agttaagtgc tataacagga 9120

gtagatgttt gtaaactcct tcgtactatt atggtaaaaa gtgcacaatg gggtagtgac 9180

cctattttgg gacaatataa ttttgaggat gaaatgacac cggaatctgt ttttaatcag 9240

gtgggtggtg ttaggttaca atcttctgtt gtaaagagag ccgcatcttg gttttggagt 9300

agatgtgtgc tagcttgttt cttgtttgtc ttgtgttcta tagttctgtt tacagctata 9360

ccatatagat attatttata tggtgctgtt gttttatttg caactgtgct ctttgtgtcg 9420

tttactgtga aacatgttat ggcatatatg gacactttcc ttttgccgac attgattacg 9480

gttattatag gagtttgtgc tgaagtacct ttcatctaca atactctaat tagtcaaatt 9540

gttattttct ttagccaatg gtatgatcca gtagtctttg acactgtagt accgtggatg 9600

tttctaccat tagtcttgta cacagctttt aaatgtatac aaggttgtta tagtgtaaat 9660

tcctttaata cttctctgtt agtgttgtat cagtttatga agttgggttt tgttatatat 9720

acctcttcta atacccttac agcctactca gaaggtaatt gggagttatt ttttgagtta 9780

gtgcacacaa ctgtgttggc taatgttagt agtaattcct taattggttt aattgtgttt 9840

aaatttgcta agtggatgct gtattattgt aatgcctcat accttaataa ttatgtccta 9900

atggctgtta ttgttaatgg cataggttgg atgtttactt gttactttgg attctattgg 9960

tggattaata aggtttttgg tttaacctta ggtaaatata gtttcaaagt ttcagtagat 10020

caatataggt atatgtgtct ccataagatt aacccaccta aaactgtgtg ggaagtcttt 10080

tctacaaata tacttataca aggtataggt ggtgaccgtg tgttgcctat agctacagtg 10140

caatccaaat tgagtgatgt aaagtgtaca actgttgtct taatgcagct tttgactaag 10200

cttaatgttg aagcaaactc aaaaatgcat gcttatcttg tagagttaca caataaaatt 10260

cttgcctcag atgatgttaa tgagtgcatg gataatttat tgggtatgct tgtaacacta 10320

ttttgtgtag atagtactat tgatttaagt gagtattgtg atgatatact taagaggtct 10380

actgttttac agtcagtcac tcaagagttt tcacacatac cttcttatgc agagtatgaa 10440

agagctaaag atctttatga aaaggtttta gctgagtcta aaaatggtag tgttacacag 10500

caagagcttg ctgcatatcg taaagctgcc aatattgcaa agtcaatctt tgatagagat 10560

ttggctgttc agaagaagtt ggatagcatg gctgaacgtg ctatgacaac tatgtataag 10620

gaagcgcgtg tcactgatag aagagctaaa cttgtttcgt cattacatgc gctattattt 10680

tctatgctta agaagataga ttctgaaaag cttaatgtac tatttgacca ggcaagtagt 10740

ggtgtcgtac cgctagctac tgttccaata gtttgtagta ataagcttac tcttgtagtg 10800

ccagatccag agacgtggat caagtgcgta gagggcatgc atgttacata ttcaacagtt 10860

gtttggaata ttgataatgt cattgatgca gatggcactg aattgcaacc aatttctaca 10920

ggtaatggtt taatttactg tataagtggt gataatatag catggcctct taaggtcaat 10980

ttgactagga atgtgcataa taaagttgat gtagtattgc agaacaacga gcttatgcct 11040

catggtgtta aaacaaaggc atgcgtggca ggtgtagatc aagcacactg cagcgtggag 11100

tctaaatgtt attatactaa tattagtggt aattcagtag tggctgctat tacctcttca 11160

aatcctaatt tgaaagttgc ctcattttta aatgaagcag gcaatcaaat ttatgttgac 11220

ttagacccac catgtaaatt tggcatgaag gtgggtgata aagtggaagt tgtttacttg 11280

tattttataa agaatacaag gtcaattgtc agaggtatgg tacttggtgc tatatctaat 11340

gtggtggtct tacagtctaa aggatatgag acagaggaag ttgatgctgt tggcatacta 11400

tcactttgtt cttttgcagt agatccggct gacacgtata ttaaatatgt ggctgcaggt 11460

aaccaacctt taggtaactg cgtaaaaatg ttgacagttc ataatggtag tggctttgct 11520

ataacatcaa agccaagccc aactcctgac caggattctt acggaggggc ttctgtgtgt 11580

ctctattgca gggcacatat agctcacccg ggaagtgcag gaaatttaga tggacgttgt 11640

caatttaaag gttcttttgt acaaatacct actacggaga aagaccctgt gggattctgt 11700

ctacgtaata aggtttgcac tgtttgtcag tgctggatag gtcatggatg tcaatgtgat 11760

tcacttagac aaccaaaacc ttcagttcaa tcagatgctg gtgcacccgg ttttgataag 11820

aattatttaa acgggtacgg ggtagcagtg aggctcggct ga 11862

<210>4

<211>7959

<212>DNA

<213> avian infectious bronchitis Virus (Infectious Bronchitis Virus)

<400>4

atgtttaaaa atttgaagcg taactgtgct cgattccaag aagtgtgtgg tactgaagat 60

ggaaatcttg agtatcgtga ctcttatttt gtggttaaac aaaccactcc tagtaattat 120

gaacatgaga aagtctgtta tgaggactta aagtcagaag taacggctga tcatgatttc 180

tttgtgttca ataagaacat ttataatatt agtaggcaac gacttactaa gtatactatg 240

atggactttt gttatgcttt gaggcatttt gacccaaagg attgcgaagt tcttaaggaa 300

atacttgtca cttatggttg tatagaagat taccatccta agtggttcga agaaaataag 360

gattggtacg atccaataga aaacccaaaa tattatgcca tgttggctaa aatgggacct 420

attgttcgtc gtgcattgtt gaatgctgtt gagtttggga acctaatggt tgagaagggt 480

tatgtaggtg tagttacact agacaaccag gacctcaatg gcaaatttta tgattttggt 540

gattttcaga aaacagcacc tggtgctggt gttcctgttt ttgatacata ttattcctac 600

atgatgccca tcatagccat gacggatgcg ttagcacctg aaaggtattt tgaatatgat 660

gtgcataagg gttacaagtc ttatgacctc ctcaagtatg attatacaga ggagaagcaa 720

gaactgtttc aaaaatactt taagtactgg gatcaagaat accaccctaa ttgtcgagat 780

tgtttcgacg acaggtgttt gatacattgt gcaaacttca acgtactttt ctctacacta 840

ataccacaaa cttcttttgg taacttatgt agaaaagtgt tcgttgatgg tgtaccattt 900

atagctactt gtggctatca ctctaaagaa ctcggtgtta ttatgaatca agataacacc 960

atgtcttttt caaaaatggg tttaagccag cttatgaaat ttgtaggaga tcctgctctt 1020

ttagtaggaa cttctaacaa tttagttgat cttagaactt cttgctttag tgtctgtgct 1080

ttggcttctg gtataataca ccagacagtc aaaccaggtc attttaataa ggatttctat 1140

gattttgctg aaaaagcagg tatgtttaag gaaggatcat caattccact taaacatttc 1200

ttctacccac agactggtaa tgccgctata aacgattatg actattatcg ttataacagg 1260

cctactatgt ttgatatacg ccagcttcta ttttgtttag aggtgacgtc taaatatttt 1320

gaatgttatg aaggtggttg tataccagct agtcaagtgg ttgttaccaa ccttgataag 1380

agtgcaggct ttccatttaa taaatttgga aaagcccgtc tctattatga aatgagtcta 1440

gaagagcagg accaactctt tgaaagtaca aagaaaaatg tcttgcctac tataactcag 1500

atgaatctta aatatgctat atccgcgaaa aatagagcgc gtactgtggc aggtgtgtcc 1560

attctttcta ctatgactaa cagacagttc catcagaagg ttcttaagtc tatagtcaat 1620

acaagaaatg cacctgtagt tattggaaca actaaatttt atggcggttg ggacaatatg 1680

ctaagaaacc tagttcaagg tgtggaagac ccaatgctta tgggttggga ctatcctaag 1740

tgtgatagag caatgccaaa tttgttgcgt atagcagcgt cattggtgct tgcccgtaaa 1800

catactaatt gttgcacatg gtctgagcgc atttatagat tgtacaatga atgcgctcaa 1860

gtattgtctg aaactgtatt agctacaggt ggtatatatg tgaaacctgg tggtactagc 1920

agtggtgatg ccaccactgc ttatgccaac agtgttttta atataataca agccacatct 1980

gctaatgttg cgcgtttact tagtgttata acgcgtgata tagtatatga tgacattaaa 2040

agcctgcagt atgagttata ccagcaggtc tataggcgag tcaattttga tcctgccttt 2100

gttgaaaagt tttattctta catgtgtaag aatttttcat taatgatctt gtctgatgat 2160

ggtgttgttt gttataacaa cacacttgcc agacaaggtc ttgtagcaga tatttctggt 2220

tttagagaaa ttctctacta ccaaaacaat gtttatatgg ccgattctaa gtgttgggtt 2280

gaatccgact tagaaaaagg cccacatgaa ttttgttcac agcatacaat gctagtagag 2340

gttgatggcg agccaagata cttgccgtat ccagacccat cacgcatttt gggtgcatgt 2400

gtatttgtag atgaagtgga taagacagaa cctgtggctg ttatggagcg ttatatcgct 2460

cttgccatag atgcttaccc gcttgtacat catgaaaacg aggagtataa gaaagttttc 2520

tttgtacttc tttcttatat tagaaagctc tatcaggagc tctcccaaag tatgcttatt 2580

gactattcct ttgttatgga tatagataag ggtagcaaat tttgggagca ggagttctat 2640

gaaaatatgt atagggctcc tacaacttta caatcttgtg gtgtttgtgt agtttgtaat 2700

agtcaaacta tactacgctg tggtaattgt attagaaaac cattcttgtg ctgtaagtgt 2760

tgctatgacc atgtcatgca cactgaccac aaaaatgttt tgtctataaa tccatacatt 2820

tgctctcaac ctggttgtgg tgaagctgat gttactaaac tttacctcgg aggtatgtct 2880

tatttctgtg gtaatcataa acctaaatta tcaataccgt tagtatctaa tggtactgtt 2940

tttggaattt acagagccaa ttgtgtgggt agcgaaaacg ttgatgattt taatcaacta 3000

gctactacta attggtctac tgttgaacct tatatattag caaatcgctg tagcgactcg 3060

ttaagacgct ttgctgctga gacagtcaaa gcaacagaag agttgcataa acagcaattt 3120

gctagtgcag aagtcagaga ggttatttct gaccgtgaat taattctatc atgggaacct 3180

ggtaagacaa ggcctccatt gaatagaaat tatgtgttta ctggctatca ctttaccagg 3240

acaagtaagg tacagctagg tgattttact tttgagaaag gtgaaggtag agatgttgtc 3300

tattataggg cgacgtctac agctaaattg tctcctggag acatttttgt cctaacatca 3360

cacaatgttg tttctcttgt agcaccaact ttgtgtcctc aacaaacttt ttccaggttt 3420

gttaatttaa gacctaatgt aatggtacca gagtgttttg ttaataacat tccattatac 3480

catttagtag gtaaacaaaa gcgtactaca gtacaaggcc ctcctggcag tggtaagtca 3540

cactttgcca taggccttgc tgcgtacttt agtaatgctc gtgtggtttt tacagcatgt 3600

tctcatgctg cagtagatgc gctttgtgaa aaagcgttta aatttcttaa agttgatgat 3660

tgcactcgca tagtacctca aaggactact gttgactgct tctcaaaatt taaagctaat 3720

gacacaggca aaaagtacat ttttagtact attaatgcct tgccagaagt tagttgtgac 3780

attctgttgg tagatgaggt tagtatgttg accaattatg aattgtcatt tattaatggt 3840

aagataaatt accagtatgt tgtgtatgta ggtgatcctg ctcaattacc ggcacctcgt 3900

actttgctca atggttcact ttcacccaag gattataatg tcatcacaaa tcttatggtt 3960

tgtgttaaac ctgacatttt ccttgcgaag tgttaccgtt gtcctaaaga aattgtagac 4020

actgtgtcta ctcttgttta tgatggaaag tttgttgcaa acaacccaga atcacgcgag 4080

tgcttcaagg ttgtagttaa taatggtaat tctgatgtag gtcacgagag tggttcagcc 4140

tacaacacaa cacaattgga atttgtgaaa gattttgttt gtcgcaataa acaatggcga 4200

gaagcaacat tcatttcacc ttataatgca atgaaccaga gagcctatcg catgcttgga 4260

cttaatgtcc agacggtaga ctcttcacaa ggttcagagt atgattatgt tatattctgt 4320

gtcacagctg attctcagca tgcattgaac attaacagat ttaatgttgc gcttacaaga 4380

gctaagcgtg gtatacttgt agtcatgcgc cagcgtgatg agttatattc tgctcttaaa 4440

tttacagagc ttgatgttga agcaagtctg caaggtacag gtttgtttaa gatttgtaac 4500

aaggaattta gtggtgtgca cccagcttat gcagtcacca ctaaagcgct tgctgcaaca 4560

tacaaggtta atgatgagct agctgcacta gttaatgtag aagctggttc agaaataact 4620

tacaagcatc ttattttttt gttagggttt aagatgagtg ttaatgttga aggttgtcac 4680

aacatgttta taacacgtga tgaggcaatt cgcaatgtaa gaggttgggt tggttttgat 4740

gtagaagcca cacatgcttg tggtactaat attggtacta accttccttt ccaagtagga 4800

ttctcaactg gtgctgactt tgttgtgacg cctgagggtc ttgttgacac ttcaataggc 4860

aataattttg agcctgtgaa ctctaaagca cctccaggtg aacaatttaa ccacttgaga 4920

gttttgttta agagtgctaa gccttggcat gttataagac caaggatagt tcagatgtta 4980

gcagacaatc tatgcaatgt ctcagattgt gtagtttttg tcacatggtg tcacggctta 5040

gaactaacta ctttgcgcta ttttgttaaa ataggcaagg aacaagtttg ttcttgtggt 5100

tctagagcta ctacttttaa ttctcatact caagcttatg cttgttggaa gcactgcttg 5160

ggttttgact ttgtttataa tccactttta gtggacgttc aacagtgggg ttactctggt 5220

aacctgcaat ttaatcatga tttgcattgt aatgtgcatg gccatgctca tgtagcttca 5280

gctgatgcta ttatgactcg ttgccttgca attaacaacg cattttgcaa agatgtcaac 5340

tgggaattaa cataccctca cattgcaaat gaggatgaag taaattccag ttgcaggtat 5400

ctacaacgca tgtatcttaa tgcgtgtgtt gatgctctta aagttaatgt tgtctatgac 5460

ataggcaacc ctaaaggtat aaaatgtgtt agacgtgggg atgtcaattt taggttttat 5520

gataagaacc caatcgtacc caatgtcaaa cagtttgagt atgactacag tcagcataaa 5580

gataagtttg ctgatggtct gtgtatgttt tggaattgta atgtggattg ttatcctgac 5640

aattctcttg tttgtaggta tgacacacgt aatttgagtg tgtttaactt accaggttgt 5700

aatggtggta gtctgtatgt taataaacat gcatttcata cacctaaatt tgatcgcatt 5760

agctttcgta atttgaaagc tatgccgttc tttttttatg actcatcgcc ttgcgaaacc 5820

attcaagttg atggagtagc gcaagacctt gtgtcattag caactaaaga ttgtatcaca 5880

aaatgcaaca ttggtggtgc tgtttgtaag aaacatgctc aaatgtatgc agaatttgtg 5940

gcttcttata atgcagctgt tacagctggt tttacttttt gggttactaa taattttaac 6000

ccttacaatt tgtggaagaa tttttcagca ctccagtcta ttgacaatat tgcttataat 6060

atgtataagg gtgggcatta cgatgctatt gcaggcgaaa tacccacagt cataactggg 6120

gataaagttt ttgttattga tcaaggtatt gaaaaggcag tttttgttaa tcaaacaact 6180

ctgcctacat cagttgcatt tgagttgtat gcgaagagaa acattcgcac actgccaaac 6240

aacagaatat tgagtggttt aggtgtagac gttacccatg gttttgtaat ttgggattac 6300

gctaaccaaa cgccactgta tcgtaatact gttaaagtat gtgcatatac ggacattgaa 6360

cctaatggtt taatagttct gtatgatgat agatgcggtg attatcaatc ttttcttgct 6420

gctgataatg ctgttttagt gtctacacag tgttataagc ggtatcctta tgtagaaata 6480

ccatcaaatc tgcttgttca gaatggtatg ccactaaaag atggagcgaa tctgtatgtt 6540

tataagagga gtaatggagc gtttgtaacg ctaccaacca cattaaacac acagggtcgc 6600

aattatgaaa cttttgagcc tcgtagtgac gtggagagag attttcttga catgtcagaa 6660

gaggacttta ttgaaaagta tggtaaggac ctaggtctac aacacatact ttatggtgaa 6720

gtagaaaaac cacaattggg tggtttacac actgttatag gaatgtacag actcttgcgc 6780

gctaataaat tagacgcgaa gtccgtaact aattcagatt ctgatgtcat gcaaaactat 6840

tttgttttgg cagacaatgg ttcttacaaa caggtttgca cagttgtaga ccttttgctt 6900

gatgacttct tagaactcct taggaacata cttaaagagt atggtactaa taagtctaaa 6960

gttgtaacag tgtccattga ttaccatagc ataaatttta tgacttggtt tgaagaaggc 7020

agtattaaaa catgttatcc acaacttcaa tcagcgtgga catgtggtta taatatgcct 7080

gaactttata aagttcagaa ttgtgttatg gaaccttgta atattccaaa ctacggtgtt 7140

ggaattacat tgcctagtgg tattatgatg aatgtggcaa agtatactca actttgccag 7200

tacctttcta aaaccacaat gtgtgtgccg cataatatgc gagtaatgca ttttggagct 7260

ggaagtgaca aaggagttgc ccctggcagc actgttttaa agcaatggct acctgaggga 7320

acacttcttg ttgataacga tattgtagat tatgtatctg atgcacatgt gtctgtgctg 7380

tcagattgca ataattataa aacagagcac aagtttgatc ttgtgatatc tgatatgtat 7440

acagataatg attcaaaaag aaaacatgaa ggcatagtag ccaataatgg taatgacgat 7500

gtcttcattt atctagctaa tttcttaaag aacaacttag ctctgggtgg tagttttgcc 7560

atcaaattaa cagagacaag ttggcatgag agtctttatg atattgcaca ggattgtgca 7620

tggtggacaa tgttttgtac agcagtaaat gcttcttctt ctgaagcatt tttactgggg 7680

attaattatt tgggtgcaag tgataatgtg aaagttagcg gaaaaacact gcacgcaaat 7740

tatatatttt ggcggaattg taattattta caaacctctg cttatagtac atttgacgtt 7800

gctaagtttg gattaaaact aaaagcaacg ccagttgtaa atttgaaaac tgaacaaaag 7860

accgacttag ttgttaatct actaaggaac ggcaagttat tgattagaga tgttggggaa 7920

gtcactgttt ttagtgacca ttttgtgtgc actatgtag 7959

<210>5

<211>1851

<212>DNA

<213> avian infectious bronchitis Virus (Infectious Bronchitis Virus)

<400>5

tctattggcc aaaatgtaac aagttgttct tatgttagtt atggcagatt ttgtattgaa 60

ccagatggtt cgttaaagat gatagtgcca gaagaattga agcagtttgt ggcaccttta 120

cttaatatta ctgaaagtgt actcatacct aacagtttta atttgactgt tacagatgag 180

tacatacaaa cacgtatgga taaggtccaa atcaattgcc ttcaatatgt ttgcggcaat 240

tctttggagt gtagaaaatt gtttcaacaa tatggtccgg tttgtgataa catattgtct 300

gttgtaaata gtgttggtca aaaagaagat atggaacttt taagtttcta ttcttctact 360

aaaccagctg gttataatgc acctgttttt agtaatatta gtactggtga ttttaatatt 420

tctcttttat taacaccacc tagtagtcct agaggtcgtt cttttataga agatttgtta 480

tttacaagtg ttgaaacagt tggtttgcca actgatgctg aatataaaaa atgcacagca 540

ggacctttag gtactcttaa ggatcttatc tgtgctaggg aatataatgg tttattagtg 600

ttgcctccaa ttattacggc tgatatgcaa acaatgtata ctgcttcttt agttggtgcc 660

atggcttttg gtggtattac agcagcaggt gccataccat ttgcaactca gatacaagcc 720

agaattaatc atttgggtat tacacagtct ctgttattga aaaatcaaga gaaaattgca 780

gcttccttta ataaggccat tggtcatatg caggaaggtt ttagaagcac ttcgctagca 840

ttacaacagg ttcaagacgt tgttaataag cagagtgcta ttcttactga aactatgaat 900

tctcttaata agaattttgg tgctatttca tcagtaattc aagacattta tgcgcaactt 960

gatgcaattc aagcagatgc acaagttgac cgtcttatta ctggcagact ctcttctctc 1020

tcagtgctag cctctgctaa gcagtctgag tacatcagag tttcccaaca acgtgaatta 1080

gccactcaaa aaattaatga gtgtgttaaa tcacaatcta ataggtatgg tttttgtggt 1140

agtggaagac atgttctttc gataccacaa aatgcaccta atggtatagt gtttatacac 1200

tttagttaca ctccagagag ttttgttaat gttactgcaa tagtaggttt ttgtgtacag 1260

cctgctaatg ctagtcagta tgcaatagtg cctgttaatg gtagaggtat ttttatacaa 1320

gttaatggta gttactatat cactgcacgt gatatgtata tgcctagaga cattactgca 1380

ggagatatag ttactcttac ttcttgtcaa gcaaattatg ttaatgtaaa taaaaccgtc 1440

attactacat ttgtggaaga tgacgatttt gattttgatt atgagttgtc gaaatggtgg 1500

aatgatacta agcatgagtt accagacttt gacgacttca attatacagt acccatactt 1560

aatattagtg gtgaaattga tcatattcaa ggtgttatac agggtcttaa tgactccctt 1620

ataaaccttg aagaactttc aatacttaaa acttatatta agtggccctg gtatgtttgg 1680

ctagccatag gctttgctat tattattttt atccttattc taggatgggt gtttttcatg 1740

acaggttgtt gtggttgttg ttgtggatgc tttggtatca ttcctctaat gagtaagtgt 1800

ggtaagaaat catcttatta cacgactttt gataatgatg tggtgactta a 1851

<210>6

<211>174

<212>DNA

<213> avian infectious bronchitis Virus (Infectious Bronchitis Virus)

<400>6

atggttcaaa ctcccgcgtc ttttgtaata ttattaattt ttctctggtt taaacttgca 60

ttaagttgtt tcagtgagtg cattgtagca cttcaacatc taatacaagt tctactccaa 120

attattaata ataatttaca atctaggctg ctcctttggc acagcctaga ctaa 174

<210>7

<211>189

<212>DNA

<213> avian infectious bronchitis Virus (Infectious Bronchitis Virus)

<400>7

atgttagatt ttgagaaaac aattgaaaca ggtgaacagt tagtacaaca aatcagtttc 60

aatttacaac atatttcaag tgttctagaa acacaaatat ttgacccatt tgagtgttgc 120

tattattcaa gtggtagttt ttatgaaata gagtcagctg acgattgttc aggtgatgaa 180

ttttattaa 189

<210>8

<211>327

<212>DNA

<213> avian infectious bronchitis Virus (Infectious Bronchitis Virus)

<400>8

atgaatttta ttaataaatc gctagaagag aacggcagtt ttctaacagc cctttatata 60

tttgtagcat ttgtagcact ttatctctta ggtagagcac tacaagcttt tgtacaagca 120

gctgatgctt gttgtttatt ttggtataca tgggtagtag tcccgggagc taagggcact 180

gcatttgtgt ataaacatac atatggtaga aaacttaaca atccggagtt agaacacgtt 240

attgttaacg agttcccgaa gaacggttgg aataataaaa ctctaccaaa ttttcaagat 300

gtccaacgag acaaattgca cccttga 327

<210>9

<211>678

<212>DNA

<213> avian infectious bronchitis Virus (Infectious Bronchitis Virus)

<400>9

atgtccaacg agacaaattg cacccttgat tctgagcagg ctattctgct cttcaaggaa 60

tataatctat ttataaccgc attcctactg tttataacca tactacttca gtatggatac 120

gcaactagga gtcggcttat ttacatactg aaaatgatag tgttatggtg tttctggccc 180

cttaacattg cagtaggtgt aatttcatgt atatacccac caaatacagg aggccttgta 240

gccgcgataa tactcactgt gtttgcgtgt ttttctttta taggttattg gattcagagc 300

attagactct ttaagcggtg tagatcttgg tggtctttta accccgagag caatgccgta 360

ggttcaatac tcctaacaaa tggtcaacaa tgtaattttg ctatagagag tgtgccaatg 420

gtactttctc ctattataaa gaatggtgct ctttattgtg agggtcagtg gctcgctaaa 480

tgtgaaccag accacttgcc taaagacatt tttgtgtgta caccagatag acgcaacatt 540

taccgtatgg tgcagaaata tactggtgac caaagcggaa ataagaaaag gtttgctaca 600

tttgtctatg caaagcagtc agtagacact ggcgagctag aaagtgtagc aacaggagga 660

agtagtcttt acacataa 678

<210>10

<211>198

<212>DNA

<213> avian infectious bronchitis Virus (Infectious Bronchitis Virus)

<400>10

atgaaatggt tgtctagttt aggacgagcg ttcacttcct gttataaagc cctactatta 60

actcaattaa gagttttaga taggttaatt ctagatcacg gaccgaggcg tactttaagt 120

tgtgccaggc gcgtgctttt attccaatta gatttagttt ataggttggt atatacgccc 180

gaccaatcgc tggtatga 198

<210>11

<211>249

<212>DNA

<213> avian infectious bronchitis Virus (Infectious Bronchitis Virus)

<400>11

atgaataata gtaaagataa tccttttcgc ggagcaatag caagaaaagc gcgaatttat 60

ctgagacaag ggttagattg tgtttacttt cttaacaaag caggacaagc agagtcttgt 120

cccgcgtgca catcactagt attccaaggg aaaacttgtg aggagcacat agataataat 180

aacttactat catggcaagc agtaaggcat ctggaaaaac agacgcccca gcgccagtta 240

ttaaactag 249

<210>12

<211>1230

<212>DNA

<213> avian infectious bronchitis Virus (Infectious Bronchitis Virus)

<400>12

atggcaagca gtaaggcatc tggaaaaaca gacgccccag cgccagttat taaactagga 60

ggaccaaaac cacccaaagt tggttcttca gggaatgcat catggtttca ggcattgaaa 120

gccaagaagc tgaatgcacc tgcacctaag tttgagggta gtggtgttcc tgataatgaa 180

aatcttaaaa caagccagca gcatgggtac tggagacgcc aagctaggta taagctaggt 240

aaaggtggaa gaaaaccagt cccagatgcg tggtacttct attacactgg aacaggacca 300

gccgctgacc tgaattgggg tgattctcag gatggtatag tgtgggttgc tgctaagggt 360

gctgatgtaa aatccagatc caatcagggt acaagagatc ctgataagtt tgatcaatac 420

ccgctacgat tttcagacgg aggacctgat ggaaatttcc gttgggactt cattcctctg 480

aatcgtggta ggagtggaag gtctacagca gcttcgtcag cagcatcctc tagggcacct 540

tcaagagaag gttctcgtgg aagacttagt ggggctgaag agaatcttat tgctcgtgca 600

gcaaaaatta ttcaagacca gcagaagaag ggctctcgca ttacaaaggc caaggcagaa 660

gagatgattc atcgccgcta ttgtaagcga acagttccac ctggtgtttc tattgataaa 720

gtttttggcc ctcgtactaa aggtaaggag ggaaattttg gtgatgacaa gatgaatgag 780

gaaggcatta aggatgggcg tgttactgca atgctcaacc ttgtacctag tagccatgct 840

tgtctttttg gtagccaagt gacacctaag cttcaacctg atggtcttca cttgactttt 900

agatttacta ctgtggtgcc acgtgatgac ccgcagtttg ataattatgt aaagatttgt 960

gatgagtgtg ttgatggtgt aggaacacgt ccaaaagacg aagttgtaag accaaaatca 1020

cgcccaagtt caagacctgc aacaagggca atttctccag cgccaagaca acagcgtcct 1080

aaaaaggaga aaaagccaaa gaagcaggat gatgaagtgg ataaagcatt gacctcagat 1140

gaggagcgga acaatgcaca gctggaattt gatgatgaac ccaaagtgat taattggggt 1200

gattcagcac ttggtgaaaa tgaactttga 1230

Claims (5)

1. An avian Infectious Bronchitis Virus (Infectious Bronchitis Virus) vaccine strain SU with a preservation number of CCTCC NO: v201648.

2. An avian infectious bronchitis vaccine composition comprising the vaccine strain SU of claim 1 and a pharmaceutically acceptable adjuvant or carrier.

3. The use of the vaccine strain SU of claim 1 in the preparation of an avian infectious bronchitis vaccine.

4. Use of the vaccine strain SU according to claim 1 for the preparation of a protein or polypeptide for use as an immunogen.

5. Use of the vaccine strain SU of claim 1 for the preparation of a protein antigen as a specific antibody for the detection of serum.

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