阅读说明：本技术 一种基因vii型新城疫病毒弱毒株及其应用 (Gene VII type Newcastle disease virus attenuated strain and application thereof ) 是由 孙化露 楚电峰 于晓璐 孙鹏 侯玉超 李振 范根成 杜元钊 于 2019-11-27 设计创作，主要内容包括：本发明提供一种自然分离的基因VII型新城疫病毒弱毒株,该毒株未进行任何基因改造,保持了该病毒作为流行毒株所具备的天然的免疫原性。本发明所提供的基因VII型弱毒株,其保藏号为：V201968。本发明分离的基因VII型新城疫病毒弱毒株NDV-VII用于制备疫苗。本发明毒株是自然分离得到的,该毒株对鸡胚最小致死量的平均致死时间为138h,1日龄雏鸡脑内接种致病指数为0.16,6周龄鸡静脉接种致病指数为0,本发明毒株具有稳定遗传性,在鸡胚上连续传代15代,其F基因并未发生致病力增强的突变。传统疫苗株LaSota株免疫保护效力比对试验证明,本发明毒株制备活疫苗免疫后抗体产生水平高于对照组,免疫后攻毒实验证明该毒株能够对鸡只提供完全保护。(The invention provides a naturally separated gene VII type Newcastle disease virus low virulent strain, which is not subjected to any gene modification, and keeps the natural immunogenicity of the virus as an epidemic virulent strain. The gene VII type low virulent strain provided by the invention has the preservation number as follows: v201968. The gene VII type newcastle disease virus low virulent strain NDV-VII separated by the invention is used for preparing vaccines. The strain is obtained by natural separation, the average lethal time of the strain to the minimum lethal dose of chick embryos is 138h, the intracerebral inoculation pathogenic index of 1-day-old chicks is 0.16, and the intravenous inoculation pathogenic index of 6-week-old chicks is 0. The traditional vaccine strain LaSota strain immune protection efficacy comparison test proves that the antibody production level of the live vaccine prepared by the strain is higher than that of a control group after immunization, and the challenge test after immunization proves that the strain can completely protect chickens.)

1. A gene VII type low virulent strain is characterized in that the preservation number of the low virulent strain is V201968.

2. Use of the attenuated strain of claim 1 in the preparation of a vaccine.

3. The use of claim 2, wherein the vaccine is a live virus vaccine.

4. A vaccine wherein the antigen comprises the attenuated strain of claim 1.

5. An F gene is characterized in that the amino acid sequence of the F gene is SEQ ID NO. 2.

6. The nucleotide fragment for encoding the F gene of claim 5, the sequence of which is SEQ ID NO 1.

7. The whole genome of the attenuated strain of claim 1, which has the nucleotide sequence of SEQ ID NO. 3.

Technical Field

The invention relates to the field of veterinary virus molecular biology and immunology, in particular to a gene VII type Newcastle disease virus low virulent strain.

Technical Field

Newcastle Disease (ND) is a highly contagious, acute virulent infectious Disease caused by Newcastle Disease Virus (NDV), which has serious harm to the world poultry industry and was once classified as a class a Disease by the international animal and Disease agency (OIE). The virus genome is single-stranded negative strand RNA, the host range is wide, the susceptibility of chickens is the highest, the morbidity and mortality of young chickens are obviously higher than those of adult chickens, and the disease can be prevalent all the year round.

NDV has only one serotype, is subjected to genetic evolution analysis by using the F gene, can be divided into two large groups of Class I and Class II, and the Class I group is divided into 18 genotypes and almost comprises all virulent strains and widely epidemic strains. The Chinese animal health and epidemiology center Wuyangong et al carries out epidemiological investigation and analysis on 535 avian NDV separated before 2010 in China, and finds that the genotype of the pandemic NDV of chicken flocks in China is mainly genotype VII. The epidemiological investigation of 8510 poultry samples in 20 provinces in China by Liuhualei and the like in 2013 shows that the NDV epidemic strain in China is mainly genotype VII. The gene analysis of Xuercong and the like at Jilin university to avian NDV isolate in the northeast region of 2013 + 2017 shows that the genotype of the main epidemic strain of the NDV is genotype VII. Furthermore, the popular NDVs in Korea and Japan around China are also mostly genotype VII. In conclusion, the method for effectively preventing and controlling the prevalence of gene VII type NDV has important significance for the purification of ND in China and the healthy development of live poultry market.

Since ND is popular, inactivated vaccine is used initially, but the vaccine has the limitations of high production cost, poor mucosal immunity and cellular immunity effects, and the share of live vaccine is continuously increased along with the separation of more and more attenuated strains. Most LaSota strains and the like mainly used for ND prevention and control in China currently are gene II type strains, the genetic distance between the LaSota strains and the gene VII type strains is large, the antigen cross protection capability is low, and complete and effective protection can not be provided for gene VII type epidemic strains of the Newcastle disease virus. Most of the separated gene VII type NDV epidemic strains are virulent strains, although antibodies can be rapidly generated, the immunized chicken is easy to generate adverse reactions such as inappetence, egg drop, virus carrying and toxin dissipating, and the like, and harm is caused to the ecological environment, so the virulent strains are not suitable for preparing vaccines, and the key for preparing the vaccines is the obtainment of gene VII type attenuated strains. In addition, the means for obtaining the attenuated strain at present is mainly to modify the virulent strain by a reverse genetic technology, and the obtained recombinant virus has the risk of returning strong toxicity after gene modification, and the toxicity is difficult to completely weaken. The natural low virulent strain is formed in the long-term natural evolution process of the virus, has good genetic stability, overcomes the defect that the toxicity of the artificially modified virulent strain is easy to return, and has better application prospect.

Disclosure of Invention

The invention aims to provide a naturally separated gene VII type newcastle disease virus attenuated strain, which is not subjected to any genetic modification, keeps the natural immunogenicity of the virus serving as an epidemic strain, can be used for preparing a gene VII type newcastle disease virus vaccine and provides a reliable gene VII type attenuated live vaccine candidate vaccine for China.

The invention provides a gene VII type Newcastle disease virus NDV-VII strain which is naturally separated and is preserved in the China center for type culture Collection in Wuhan university in 2019, 10, 15 and the preservation number is CCTCC NO: V201968.

The gene VII type Newcastle disease virus low virulent strain NDV-VII separated by the invention is used for preparing a vaccine;

the vaccine is preferably a live vaccine.

In another aspect, the invention provides a vaccine, wherein the antigen is the gene VII type Newcastle disease virus low virulent strain NDV-VII strain.

The invention also provides the F gene of the NDV-VII strain, the amino acid sequence of the F gene is SEQ ID NO. 2, the nucleotide sequence of one coding gene is SEQ ID NO. 1,

the invention also provides a whole genome of the NDV-VII strain, and the nucleotide sequence of the whole genome is SEQ ID NO. 3.

The strain is obtained by natural separation, the average lethal time (MDT) of the strain to the minimum lethal dose of chick embryos is 138h, the intracerebral inoculation pathogenic index (ICPI) of 1-day-old chicks is 0.16, and the intravenous inoculation pathogenic index (IVPI) of 6-week-old chicken wings is 0. The traditional vaccine strain LaSota strain immune protection efficacy comparison test proves that the antibody production level of the live vaccine prepared by the strain is higher than that of a control group after immunization, and the challenge test after immunization proves that the strain can completely protect chickens.

Drawings

FIG. 1: NDV-VII strain F fragment PCR identification result;

FIG. 2: PCR amplification electrophoretogram of 9 segments of the whole genome of the NDV-VII strain;

FIG. 3: and constructing a phylogenetic tree for the F fragment.

Detailed Description

The invention relates to a biological material: the LaSota live vaccine is provided by Qingdao Yibang bioengineering Co.Ltd; the gene NVII type virulent strain JS06/02 is preserved by the company; standard positive sera of H5, H7, H9 subtypes AI and ND were purchased from Chinese veterinary medicine inspection institute.