阅读说明：本技术 一株禽传染性支气管炎病毒的天然弱毒株及其应用 (Natural low virulent strain of avian infectious bronchitis virus and application thereof ) 是由 秦爱建 耿仁浩 周海生 钱琨 邵红霞 叶建强 于 2019-09-06 设计创作，主要内容包括：本发明公开了一株禽传染性支气管炎病毒(IBV)的天然弱毒株(CK/CH/2014/QL1403)。该IBV毒株不引起SPF鸡发病,基因分析属于一个新型IBV分支,该IBV分离株CK/CH/2014/QL1403可以用鸡胚增殖传代,鸡胚尿囊液繁殖的IBV稀释后免疫鸡,可以提供免疫鸡抵抗禽传染性支气管炎病毒强毒的攻击,可以用作疫苗研究。(The invention discloses a natural low virulent strain (CK/CH/2014/QL1403) of avian Infectious Bronchitis Virus (IBV). The IBV strain does not cause the incidence of SPF (specific pathogen free) chickens, gene analysis belongs to a novel IBV branch, the IBV isolate CK/CH/2014/QL1403 can be used for breeding passages by chick embryos, and the IBV bred by chick embryo allantoic fluid can be used for immunizing chickens after being diluted, so that the IBV strain can provide the immunized chickens with resistance to the attack of virulent avian infectious bronchitis viruses, and can be used for vaccine research.)

1. A natural low virulent strain of avian infectious bronchitis virus is characterized in that the natural low virulent strain of avian infectious bronchitis virus is preserved in a China center for type culture collection with the preservation date of 2019, 1 month and 27 days and the preservation number of CCTCC NO: V201907, and is named as avian infectious bronchitis virus QL1403 by classification.

2. The natural low virulent strain of avian infectious bronchitis virus according to claim 1, wherein the gene fragment at position 24840 of the 20395 th ~ th in the genome of the natural low virulent strain is a natural recombinant gene fragment.

3. Use of the natural low virulent strain of avian infectious bronchitis virus according to claim 1 or 2 for the preparation of a medicament for preventing or treating avian infectious bronchitis.

4. The use according to claim 3, wherein the vaccine candidate is in the form of one of an injection, a spray, and a drop.

5. A vaccine for preventing avian infectious bronchitis, which comprises a natural low virulent strain of the avian infectious bronchitis virus according to claim 1.

6. The vaccine for preventing avian infectious bronchitis according to claim 5, characterized in that the avian infectious bronchitis is avian infectious bronchitis.

Technical Field

The invention belongs to the technical field of biology, and relates to a natural low virulent strain of avian infectious bronchitis virus and application thereof.

Background

Avian Infectious Bronchitis (IB) is an acute, highly contagious disease caused by avian Infectious Bronchitis Virus (IBV). After IBV infection, clinical symptoms and pathological changes are mainly concentrated in respiratory tract, kidney and reproductive tract, so that the meat-feed ratio of poultry is reduced, the egg yield and egg quality are reduced, the mortality rate is increased after secondary infection, and huge economic loss is brought to poultry industry in China. IBV was first isolated and reported in China in the 80 th century of the 20 th century and then widely prevalent in China. The pathogeny of the disease has a plurality of serotypes, different serotype strains have poor cross protection effect, although IBV vaccines (Mass, 4/91 and tl/CH/LDT3/03 vaccines) are widely used, IB still frequently explodes, new serotypes, genotypes or variant strains continuously appear, and the control of the IB is greatly challenged.

The QX strain was first reported in 1997 in our country and subsequently became prevalent in many countries including asia, europe and africa. The virus of this type was first reported to cause predominantly gastro-glandular lesions in the QXIBV strain, belonging to the gastro-glandular type. But recent reports show that the strain is mainly caused by kidney and respiratory tract diseases and can cause death of chicks. More than 50% of isolated IBV strains in China between 1995 and 2009 are reported to belong to QX type IBV, and the proportion is probably higher in the southern part of China. Mass type attenuated vaccines 4/91 and tl/CH/LDT3/03, such as H120, H52, 4/91 and LDT3-A, are used in IB epidemic areas, even in combination in part of areas, but after immunization, the IBV of the type still frequently explodes and is reported to be separated, and the Mass type attenuated vaccine H120 is reported in documents to be incapable of preventing the virus of the type. In conclusion, commercial vaccines frequently used in China at present cannot effectively prevent the diseases of the QX-type IBV.

In order to control the prevalence and the occurrence of the disease, the disease is separated, identified and sequenced by pathogenic chicken, and relevant animal immunity tests are carried out, so that a better effect is achieved.

Disclosure of Invention

The purpose of the invention is as follows: the invention provides an IBV isolated strain which can be used as a vaccine based on the isolation and identification of IBV (CK/CH/2014/QL1403) by the inventor. The virus is recently isolated in the course of routine pathogen monitoring in a chicken flock in China and kept in the laboratory of the department of cooperative education of preventive medicine provinces of poultry. The isolated enveloped single-stranded RNA virus belongs to the order Nidovirales order Coronaviridae (Coronavir). The natural low virulent strain of the avian infectious bronchitis virus is preserved in the China center for type culture collection, the preservation date is 2019, 1 month and 27 days, and the preservation number is CCTCC NO: v201907, which is classified and named as avian infectious bronchitis virus QL 1403. The invention relates to an isolated avian infectious bronchitis virus related to a known member of the family coronaviridae.

The invention aims to solve the technical problem of providing the application of the natural low virulent strain of the avian infectious bronchitis virus in the preparation of the medicine for preventing or treating the avian infectious bronchitis.

The technical problem to be solved by the invention is to provide the drop for preventing or treating the avian infectious bronchitis.

The invention finally solves the technical problem of providing the vaccine for preventing the avian infectious bronchitis.

The technical scheme is as follows: in order to achieve the technical purpose, the invention adopts the following technical scheme: a natural low virulent strain of avian infectious bronchitis virus is preserved in China Center for Type Culture Collection (CCTCC) with the preservation date of 2019, 1 month and 27 days and the preservation number of CCTCC NO: v201907, which is classified and named as avian infectious bronchitis virus QL 1403.

Wherein, the genome 20395-24840 segment of the natural low-virulent strain of CK/CH/2014/QL1403 is found to be recombined by applying the analysis of recombination technology. Further, the analysis of the recombination source of the genome sequence shows that CK/CH/2014/QL1403 is derived from natural recombination of a QX and TC07-2 strain, and the strain obtained through analysis of the evolutionary tree belongs to a novel IBV strain.

The invention also comprises the application of the natural low virulent strain of the avian infectious bronchitis virus in the preparation of vaccines for preventing avian infectious bronchitis.

The invention also provides a vaccine for preventing the avian infectious bronchitis, which comprises the natural low virulent strain of the avian infectious bronchitis virus.

Wherein the avian infectious bronchitis is chicken infectious bronchitis.

Compared with the prior art, the invention has the following advantages and beneficial effects:

1. The invention relates to a natural low virulent strain of avian infectious bronchitis virus

IBVCK/CH/2014/QL1403, and animal experiments show that the strain does not cause SPF chicken to generate clinical symptoms and pathological changes, and belongs to a natural low-virulent strain.

2. The IBV vaccine is prepared by inoculating a natural low virulent strain IBVCK/CH/2014/QL1403 of the separated avian infectious bronchitis virus to SPF chick embryos, and then collecting the SPF chick embryos. After the SPF chickens are immunized for 14 days, the IBV antibody reaction of the chicken flock is positive; after immunizing SPF chickens for 28 days, the new vaccine-receiving flock was challenged with a wild strain of QX genotype IBV. The novel vaccine provides sufficient antibody titers against the QX genotype IBV and provides effective protection for the flock.

drawings

FIG. 1 shows the result of the evolutionary tree analysis of the IBVCK/CH/2014/QL1403S1 gene (GenBank accession number KU 361188). Analysis of the S1 gene evolutionary tree shows that CK/CH/2014/QL1403 and CK/CH/IBTZ/2012(GenBank accession number KF663559) and CK/CH/SD09/005(GenBank accession number KF668605) strains belong to the same gene branch and belong to IBV novel strains, the phylogenetic tree is constructed by the adjacency method, and the self-guide value is deduced from 1000 repeats.

FIG. 2A shows the results of the site homology analysis of the genomic sequence IBVCK/CH/2014/QL1403 (GenBank accession number KU 361188). The homology of reference strain CK/CH/SD09/005(GenBank accession KF668605) and CK/CH/2014/QL1403 in the genomic sequence site is shown in red line; the homology of reference strain SDIB821/2012(GenBank accession KF574761) to CK/CH/2014/QL1403 genomic sequence site is shown in blue line; the homology of the reference strain M41(GenBank accession number DQ834384) to the CK/CH/2014/QL1403 genomic sequence site is shown in grey line.

FIG. 2B shows the results of analysis of the different fragment evolutionary trees of the genomic sequence IBV CK/CH/2014/QL1403 (GenBank accession number KU 361188). The evolutionary tree analysis shows that CK/CH/2014/QL1403 genome sequence fragments 1 to 20394 and 24841 to 27691 belong to a gene branch with SDIB821/2012(GenBank retrieval number KF574761) and YX10(GenBank retrieval number JX840411) strains and belong to IBV QX type strains; the CK/CH/2014/QL1403 genome sequence fragments 20395-24840 belong to a gene branch, and the CK/CH/IBTZ/2012(GenBank retrieval number KF663559) and CK/CH/SD09/005(GenBank retrieval number KF668605) strains belong to a novel IBV strain. The system tree is constructed by the adjacency method. The bootstrap values were inferred from 1000 repetitions.

Detailed Description

The following detailed description of the preparation process and application process of the product of the present invention will be provided by the applicant in conjunction with specific examples to facilitate the clear understanding of the present invention by those skilled in the art. It should be understood that the following examples should not be construed as limiting the scope of the claims of the present application in any way.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

IBV isolate CK/CH/2014/QL1403 was isolated from a chicken flock of Jiangsu. The specific separation method is as follows: collecting trachea, lung, kidney, oviduct, etc. of the clinic chicken. Preparing a tissue sample into 10% w/v homogenate by PBS (containing 10000U of penicillin and 100 mu g of streptomycin, pH7.2), centrifuging for 10min at 4 ℃, 5000 Xg, taking the supernatant, placing the supernatant in a 37 ℃ incubator for 20min, inoculating 9-11-day SPF (specific pathogen free) chick embryos in each processed sample through a chorioallantoic cavity, culturing and observing for 120h at 37 ℃, discarding dead chick embryos within 24h, aseptically collecting allantoic fluid, and observing characteristic lesions after IBV infection after 3 blind generations. The IBV isolate CK/CH/2014/QL1403 obtained by separation is preserved in the China center for type culture Collection with the preservation date of 2019, 1 month and 27 days and the preservation number of CCTCC NO: v201907, which is classified and named QL 1403.

Chick embryos infected with the virus develop poorly, the embryo bodies are hemiplegic and contracture, and kidney urate deposits. Generally, chick embryo lesions are not evident after primary inoculation.

random Hexamer was purchased from Huada.

RiboLock RNase Inhibitor was purchased from Thermo with product catalog number EO 0381.

M-MLV reverse transcriptase was purchased from TaKaRa under catalog number D2639A.

LATaq DNA polymerase was purchased from TaKaRa under catalog number RR 25A.

The 5' -Full RACE kit was purchased from TaKaRa under catalog No. D315.

the 3' -Full RACE kit was purchased from TaKaRa under catalog No. D314.

SPF chick embryos were purchased from Merria, France.