CN106754746B - Avian infectious bronchitis virus vaccine strain, vaccine and application thereof - Google Patents
Avian infectious bronchitis virus vaccine strain, vaccine and application thereof Download PDFInfo
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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
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 TakaraTMII 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.
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 105.0EID50Animals regression experiments using vaccine strains H120, L DT3 eye drops of nasal challenge as positive controls, 103.5EID50And/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 105.0EID50. 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 105.0EID50. 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 105.5EID50。
(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 103.5EID50、104.5EID50、105.5EID50Setting commercial vaccine strain L DT3 and H120 positive control group, and the immune dose is a recommended dose (10) of one feather3.5EID50) 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 use5.5The 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 104.5EID50、105.5EID50、106.5EID50Feather, 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 105.5EID50Immunization of the feather. 21 days after immunization, all groups used the corresponding challenge strain (parental strain SF or heterologous strain M41) at 105.0EID50The 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 105.0EID50After 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 ways104.5EID50Feather, 105.5EID50Feather sum 106.5EID50Immunization 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 105.0EID50The 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
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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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CN110358740B (en) * | 2019-08-12 | 2022-07-22 | 江苏农牧科技职业学院 | QX type avian infectious bronchitis virus strain and application thereof |
Citations (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN101514334A (en) * | 2009-03-23 | 2009-08-26 | 中国农业科学院哈尔滨兽医研究所 | Chicken infectivity bronchitis virus attenuated vaccine strain and application thereof |
CN103007274A (en) * | 2012-12-31 | 2013-04-03 | 青岛易邦生物工程有限公司 | Bivalent live vaccine for infectious chicken bronchitis |
CN103497934A (en) * | 2013-10-08 | 2014-01-08 | 南京天邦生物科技有限公司 | Avian infectious bronchitis virus vaccine strain (HF2 strain) and application thereof |
CN104353070A (en) * | 2014-11-05 | 2015-02-18 | 中山大学 | Genetic engineering subunit vaccine of chicken infectious bronchitis virus and preparation method thereof |
CN104383529A (en) * | 2014-11-28 | 2015-03-04 | 中山大学 | High-efficiency chicken infectious bronchitis inactivated vaccine and preparation method thereof |
CN105671003A (en) * | 2016-03-18 | 2016-06-15 | 华南农业大学 | Infectious bronchitis low-virulent live vaccine YX10 D90 strain |
CN105802918A (en) * | 2014-12-29 | 2016-07-27 | 普莱柯生物工程股份有限公司 | Avian nephropathogenic infectious bronchitis virus strain as well as vaccine composition, preparation method and application thereof |
-
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Patent Citations (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN101514334A (en) * | 2009-03-23 | 2009-08-26 | 中国农业科学院哈尔滨兽医研究所 | Chicken infectivity bronchitis virus attenuated vaccine strain and application thereof |
CN103007274A (en) * | 2012-12-31 | 2013-04-03 | 青岛易邦生物工程有限公司 | Bivalent live vaccine for infectious chicken bronchitis |
CN103497934A (en) * | 2013-10-08 | 2014-01-08 | 南京天邦生物科技有限公司 | Avian infectious bronchitis virus vaccine strain (HF2 strain) and application thereof |
CN104353070A (en) * | 2014-11-05 | 2015-02-18 | 中山大学 | Genetic engineering subunit vaccine of chicken infectious bronchitis virus and preparation method thereof |
CN104383529A (en) * | 2014-11-28 | 2015-03-04 | 中山大学 | High-efficiency chicken infectious bronchitis inactivated vaccine and preparation method thereof |
CN105802918A (en) * | 2014-12-29 | 2016-07-27 | 普莱柯生物工程股份有限公司 | Avian nephropathogenic infectious bronchitis virus strain as well as vaccine composition, preparation method and application thereof |
CN105671003A (en) * | 2016-03-18 | 2016-06-15 | 华南农业大学 | Infectious bronchitis low-virulent live vaccine YX10 D90 strain |
Non-Patent Citations (3)
Title |
---|
Development and efficacy of a novel live-attenuated QX-likenephropathogenic infectious bronchitis virus vaccine in China;Keyu Feng等;《vaccine》;20150127;第33卷;第1113-1120页 * |
QX 型传染性支气管炎弱毒疫苗致弱机制研究及其安全性与免疫效力的初步评价;霍亚飞;《中国优秀硕士学位论文全文数据库 农业科技辑》;20160415(第4期);摘要 * |
QX型鸡传染性支气管炎弱毒疫苗的研制;陈启稳;《中国优秀硕士学位论文全文数据库 农业科技辑》》;20150115(第1期);摘要 * |
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