WO2021103421A1 - Gene vii type newcastle disease virus attenuated strain and use thereof - Google Patents

Gene vii type newcastle disease virus attenuated strain and use thereof Download PDF

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WO2021103421A1
WO2021103421A1 PCT/CN2020/088921 CN2020088921W WO2021103421A1 WO 2021103421 A1 WO2021103421 A1 WO 2021103421A1 CN 2020088921 W CN2020088921 W CN 2020088921W WO 2021103421 A1 WO2021103421 A1 WO 2021103421A1
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strain
attenuated strain
ndv
vaccine
vii
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孙化露
楚电峰
于晓璐
孙鹏
侯玉超
李振
范根成
杜元钊
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青岛易邦生物工程有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
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    • C12N2760/18011—Paramyxoviridae
    • C12N2760/18111—Avulavirus, e.g. Newcastle disease virus
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    • C12N2760/00011—Details
    • C12N2760/18011—Paramyxoviridae
    • C12N2760/18111—Avulavirus, e.g. Newcastle disease virus
    • C12N2760/18134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
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Definitions

  • This application relates to the field of molecular biology and immunology of veterinary viruses, in particular to an attenuated strain of genotype VII Newcastle disease virus and its application.
  • Newcastle Disease is a highly contagious, acute and severe infectious disease caused by Newcastle Disease Virus (NDV), which is a serious hazard to the world's poultry industry.
  • the virus genome is a single-stranded negative-strand RNA with a wide host range, among which chickens are the most susceptible, and the morbidity and mortality of young chickens are significantly higher than that of adult chickens. The disease can be prevalent throughout the year. NDV has only one serotype.
  • the genetic evolution analysis using F gene can be divided into two major groups, Class I and Class II. The Class I group is divided into 18 genotypes, which contain almost all virulent strains and widely circulating strains.
  • Vaccines are the most economical and effective means to prevent and control infectious diseases. Since the ND epidemic, inactivated vaccines were first used, which have the limitations of high production costs, poor mucosal immunity and cellular immunity. With the isolation of more and more attenuated strains, the share of live vaccines continues to increase. At present, the LaSota strains mainly used for ND prevention and control in China are mostly genotype II strains, which are genetically distant from genotype VII strains and have low antigen cross-protection. They cannot provide complete coverage for the newcastle disease virus genotype VII epidemic strains. Effective protection.
  • genotype VII NDV isolated in the prior art are often virulent strains, although they can quickly produce antibodies, immunized chickens are prone to poor appetite, decreased egg production, poisoning, and other adverse reactions, which are harmful to the ecological environment. Cause harm, so obtaining the attenuated genotype VII strain is the key to vaccine preparation.
  • the method of obtaining attenuated strains in the prior art is mainly to transform the virulent strains through reverse genetic technology, and the obtained recombinant virus has the disadvantages of genetic modification, such as the risk of returning to strong virulence, and it is difficult to completely weaken the virulence.
  • This application provides an attenuated strain of genotype VII Newcastle disease virus and its application.
  • the strain is naturally isolated without any genetic modification, and maintains the natural immunogenicity possessed by the virus as an epidemic strain.
  • the first embodiment of this application provides an attenuated strain of genotype VII Newcastle disease virus, which may also be referred to as: NDV-VII strain, which was deposited on October 15, 2019 in the Chinese Type Culture Collection at Wuhan University Center (CCTCC), its deposit number is CCTCC V201968.
  • NDV-VII strain which was deposited on October 15, 2019 in the Chinese Type Culture Collection at Wuhan University Center (CCTCC), its deposit number is CCTCC V201968.
  • the second embodiment of the present application provides an application of the attenuated strain.
  • the isolated attenuated strain of Newcastle Disease Virus (NDV-VII strain) of gene type VII can be used to prepare a vaccine.
  • the vaccine is a live vaccine.
  • the third embodiment of the present application provides a vaccine, wherein the antigen is the aforementioned attenuated strain of genotype VII Newcastle disease virus, that is, the NDV-VII strain.
  • the fourth embodiment of the present application provides the F gene of the NDV-VII strain, the nucleotide sequence of which is SEQ ID No. 1; the amino acid sequence of the protein encoded by the nucleotide sequence is SEQ ID No. 2.
  • the fifth embodiment of the present application provides the entire genome of the NDV-VII strain, and its nucleotide sequence is SEQ ID No. 3.
  • the attenuated strain has passed through 20 consecutive generations of chicken embryos, and its MDT is greater than 120h; there is no mutation in the amino acid sequence of the cleavage site of the F gene.
  • the NDV-VII strain in this application was obtained by natural isolation.
  • the average lethal time (MDT) of this strain to chicken embryos was 138h, and the intracerebral inoculation pathogenicity index (ICPI) of 1-day-old chicks was 0.16.
  • the IVPI of 6-week-old chicken wings intravenously was 0.
  • the strain has stable heredity and has been successively passaged on chicken embryos for 20 generations, and its F gene does not have mutations with enhanced pathogenicity.
  • the comparison test of the immunoprotective efficacy of the traditional vaccine strain LaSota strain proved that the antibody production level of the live vaccine prepared by this strain was higher than that of the control group.
  • the post-immunization challenge experiment proved that the strain can provide complete protection to chickens.
  • Figure 1 PCR identification result of F fragment of NDV-VII strain
  • Figure 2 PCR amplification electrophoresis diagram of 9 fragments of the whole genome of NDV-VII strain
  • Figure 3 Phylogenetic development tree constructed based on F gene.
  • LaSota live vaccine and genotype VII Newcastle disease virus virulent strain JS02/06 were provided by Qingdao Yibang Bioengineering Co., Ltd.; H5, H7, H9 subtype AI and ND standard positive sera were all provided Purchased from China Veterinary Drug Inspection Institute.
  • Example 1 Screening of attenuated strains of NDV and determination of MDT, ICPI, IVPI
  • NDV-VII strain a suspected NDV strain was isolated from chickens submitted for clinical inspection in a certain area of Shandong, and was named NDV-VII strain. Allantoic fluid was collected by inoculating chicken embryos, and HA titer was determined. After 20 consecutive passages, the HA titer determination results showed that the HA titer of the chicken embryo allantoic fluid of this strain of virus stabilized at 9 log2 after 4 generations in the chicken embryo. The harvested allantoic fluid was subjected to a micro-hemagglutination inhibition test (HI). The results showed that the allantoic fluid sample only reacted with ND standard positive sera and negatively reacted with H5, H7, and H9 positive sera.
  • HI micro-hemagglutination inhibition test
  • tissue samples of healthy chickens to be tested were sent from a farm in Shandong in April 2019.
  • the trachea, liver, heart, intestine and other tissues of the chicken were taken, and the four antibodies PBS (Penicilin 10000IU/mL, pH 7.2) were added.
  • Centrifuge at 8000r/min for 10min take 0.2mL of supernatant and inoculate it in 9-10 day-old SPF chicken embryos, discard the chicken embryos that died within 24h, collect the allantoic fluid of the chicken embryos after 96h, and determine the HA titer.
  • the effective value of the allantoic fluid was diluted twice and then inoculated with chicken embryo fibroblasts (CEF) for plaque cloning and purification three times; the chicken embryo was again inoculated, and the chicken embryo allantoic fluid was collected 96 hours later, and the HA titer was measured.
  • the virus strain is purified, it is passaged in chicken embryos for 4 generations, and the HA titer of allantoic fluid can be stabilized at 9log2; the virus strain is obtained and stored.
  • the harvested allantoic fluid was subjected to a micro-hemagglutination inhibition test (HI). The results showed that the allantoic fluid sample only reacted with ND standard positive sera and negatively reacted with H5, H7, and H9 positive sera.
  • MDT is called Mean death time
  • ICPI is called Intracephalic pathogenicity index
  • IVPI is called Intravenous pathogenicity index.
  • MDT refers to the average time for the death of chicken embryos due to the minimum lethal dose. Among them, MDT less than 60h is a virulent strain of NDV, MDT value between 60h and 90h is a moderately virulent strain, and MDT greater than 90h is a weakly virulent strain.
  • the MDT calculation formula is:
  • ICPI intracerebral pathogenicity index
  • IVPI intravenous inoculation pathogenicity index
  • Tables 1 to 3 show that the MDT of the NDV-VII strain is 138h, the ICPI is 0.16, and the IVPI is 0, which conforms to the attenuated characteristics of NDV, which proves that the NDV-VII strain isolated this time is an attenuated strain.
  • NDV-VII strain of the attenuated genotype VII Newcastle disease virus obtained in this screening has been deposited at Wuhan University on October 15, 2019 in accordance with the Budapest Treaty on the International Recognition of the Deposit of Microorganisms Used in Patent Procedures.
  • the Culture Collection, its deposit number is CCTCC V201968.
  • the primers were designed according to the F gene sequence of NDV published by NCBI. Upstream primer: NDV F-F: ATGGGCTCCAAACCTTCTACCAG; downstream primer: NDV F-R: AAACTGCTGCATCTTCCCAACCG.
  • the F fragment of the NDV-VII strain was amplified and identified, and the target fragment was about 550 bp.
  • the nucleotide sequence of the F gene fragment is SEQ ID No. 1
  • the amino acid sequence of the nucleotide sequence encoding the protein is SEQ ID No. 2.
  • the F gene has a full length of 1662 bp, and the amino acid sequence at the cleavage site is 112 GRQGRL 117 , which conforms to the characteristics of the attenuated strain cleavage site.
  • the NDV-VII strain has a full-length genome of 15186bp, which is inconsistent with the conventionally isolated gene type VII NDV genome (15192bp). It is speculated that the NDV-VII strain has undergone recombination during the long-term clinical epidemic. And finally stable inheritance.
  • the amino acid characteristics at the cleavage site of the F gene of the virus are closely related to virulence. According to the whole genome sequence, the amino acid sequence at the cleavage site of the F gene of this strain is 112 GRQGRL 117 , which is consistent with the general characteristics of the attenuated strain’s cleavage site. This indicates that the isolate NDV-VII has the characteristics of attenuating toxicity.
  • the NDV-VII strain was passed through chicken embryos for serial passage experiments, virus MDT and ICPI were measured every 5 generations, and the F gene was sequenced, and the amino acid sequence of the cleavage site was analyzed to pass to 20 generations.
  • the results showed that after 20 consecutive generations of chicken embryos, the MDT of the strain was always greater than 120h, and there was no mutation in the amino acid sequence at the cleavage site of the F gene, which proved that although the strain may be a naturally recombined strain, its genetic performance is stable and suitable as a vaccine Candidate strains (Table 5).
  • Example 6 Preparation, safety test and sterility test of NDV-VII strain live vaccine
  • SPF chickens aged 21 days were randomly divided into 3 groups, each with 10 chickens.
  • the first group is the NDV-VII strain freeze-dried live vaccine group
  • the second group is the LaSota strain live vaccine group
  • the third group For the PBS control group, immunized with nasal drops and eye drops, 0.1 mL/moist. After immunization, each group was reared in isolation, and blood was collected at 14 days, 21 days, and 28 days after immunization to determine antibodies. After 28 days of blood collection, challenge with genotype VII virulent strain JS 02/06.
  • the method of challenge is nasal drops and eye drops at a dose of 10 6 EID 50 per bird. The animals are raised in an isolator.
  • Table 9 Virus isolation results on the 3rd and 5th days after challenge
  • the NDV-VII strain constructed in this application has good immunogenicity. It was found that the vaccine is safe for chickens, can induce the production of higher neutralizing antibodies, and can protect animals.
  • the currently popular Genotype VII virulent strain has a very good protective effect and has broad application prospects.

Abstract

Provided are a naturally isolated gene VII type Newcastle disease virus attenuated strain and the use thereof, wherein the deposit number thereof is CCTCC V201968. The strain has a stable inheritance, is prepared into a live vaccine to immunize chickens, can resist an attack from a gene VII type virulent strain and can be used for preparing a vaccine.

Description

基因VII型新城疫病毒弱毒株及其应用Genotype VII Newcastle Disease Virus Attenuated Strain and Its Application
本申请要求在2019年11月27日提交中国专利局、申请号为201911182037.X、发明名称为“一种基因VII型新城疫病毒弱毒株及其应用”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。This application claims the priority of a Chinese patent application filed with the Chinese Patent Office on November 27, 2019, the application number is 201911182037.X, and the invention title is "A Genotype VII Newcastle Disease Virus Attenuated Strain and Its Application", all of which The content is incorporated in this application by reference.
技术领域Technical field
本申请涉及兽医病毒分子生物学与免疫学领域，具体涉及一株基因VII型新城疫病毒弱毒株及其应用。This application relates to the field of molecular biology and immunology of veterinary viruses, in particular to an attenuated strain of genotype VII Newcastle disease virus and its application.
技术背景technical background
新城疫(Newcastle Disease，ND)是由新城疫病毒(Newcastle Disease Virus，NDV)引起的一种高度接触性、急性烈性传染病，对世界养禽业危害严重。该病毒基因组为单股负链RNA，宿主范围广，其中以鸡的易感性最高，且幼龄鸡的发病率和死亡率明显高于成年鸡，该病一年四季均可流行。NDV只有一个血清型，利用F基因进行遗传进化分析，可分为Class I和ClassⅡ两大类群，Class I类群分为18个基因型，几乎包含所有强毒株及广泛流行毒株。Newcastle Disease (ND) is a highly contagious, acute and severe infectious disease caused by Newcastle Disease Virus (NDV), which is a serious hazard to the world's poultry industry. The virus genome is a single-stranded negative-strand RNA with a wide host range, among which chickens are the most susceptible, and the morbidity and mortality of young chickens are significantly higher than that of adult chickens. The disease can be prevalent throughout the year. NDV has only one serotype. The genetic evolution analysis using F gene can be divided into two major groups, Class I and Class II. The Class I group is divided into 18 genotypes, which contain almost all virulent strains and widely circulating strains.
疫苗是防控传染病最经济有效的手段，自ND流行以来，最初使用灭活疫苗，其具有生产成本高，粘膜免疫及细胞免疫效果差等局限。随着越来越多弱毒株的分离，活疫苗所占份额持续增加。当前中国对ND防控主要使用的LaSota株等多为基因II型毒株，与基因VII型毒株遗传距离较大，抗原交叉保护力较低，不能对新城疫病毒基因VII型流行株提供完全有效的保护。由于现有技术中分离到的基因VII型NDV流行毒株常为强毒株，虽然能够迅速产生抗体，但是免疫鸡易产生食欲不振、产蛋下降、带毒散毒等不良反应，对生态环境造成危害，因此获得基因VII型弱毒株是疫苗制备的关键。现有技术中获得弱毒株的手段主要是通过反向遗传技术对强毒株进行改造， 获得的重组毒经过基因改造，有毒力返强的风险，且毒力很难完全致弱等缺点。Vaccines are the most economical and effective means to prevent and control infectious diseases. Since the ND epidemic, inactivated vaccines were first used, which have the limitations of high production costs, poor mucosal immunity and cellular immunity. With the isolation of more and more attenuated strains, the share of live vaccines continues to increase. At present, the LaSota strains mainly used for ND prevention and control in China are mostly genotype II strains, which are genetically distant from genotype VII strains and have low antigen cross-protection. They cannot provide complete coverage for the newcastle disease virus genotype VII epidemic strains. Effective protection. Since the epidemic strains of genotype VII NDV isolated in the prior art are often virulent strains, although they can quickly produce antibodies, immunized chickens are prone to poor appetite, decreased egg production, poisoning, and other adverse reactions, which are harmful to the ecological environment. Cause harm, so obtaining the attenuated genotype VII strain is the key to vaccine preparation. The method of obtaining attenuated strains in the prior art is mainly to transform the virulent strains through reverse genetic technology, and the obtained recombinant virus has the disadvantages of genetic modification, such as the risk of returning to strong virulence, and it is difficult to completely weaken the virulence.
发明内容Summary of the invention
本申请提供了一种基因VII型新城疫病毒弱毒株及其应用，该毒株自然分离而得，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。This application provides an attenuated strain of genotype VII Newcastle disease virus and its application. The strain is naturally isolated without any genetic modification, and maintains the natural immunogenicity possessed by the virus as an epidemic strain.
本申请的第一种实施方式提供了一种基因VII型新城疫病毒弱毒株，以下也可简称为：NDV-VII株，于2019年10月15日保藏在位于武汉大学的中国典型培养物保藏中心(CCTCC)，其保藏号为CCTCC V201968。The first embodiment of this application provides an attenuated strain of genotype VII Newcastle disease virus, which may also be referred to as: NDV-VII strain, which was deposited on October 15, 2019 in the Chinese Type Culture Collection at Wuhan University Center (CCTCC), its deposit number is CCTCC V201968.
本申请的第二种实施方式提供了一种所述弱毒株的应用，具体地，所分离的基因VII型新城疫病毒弱毒株(NDV-VII株)可用于制备疫苗。The second embodiment of the present application provides an application of the attenuated strain. Specifically, the isolated attenuated strain of Newcastle Disease Virus (NDV-VII strain) of gene type VII can be used to prepare a vaccine.
可选地，所述的疫苗为活疫苗。Optionally, the vaccine is a live vaccine.
本申请的第三种实施方式提供了一种疫苗，其中抗原为上述的基因VII型新城疫病毒弱毒株，即NDV-VII株。The third embodiment of the present application provides a vaccine, wherein the antigen is the aforementioned attenuated strain of genotype VII Newcastle disease virus, that is, the NDV-VII strain.
本申请的第四种实施方式提供了NDV-VII株的F基因，其核苷酸序列为SEQ ID No.1；所述核苷酸序列所编码的蛋白的氨基酸序列为SEQ ID No.2。The fourth embodiment of the present application provides the F gene of the NDV-VII strain, the nucleotide sequence of which is SEQ ID No. 1; the amino acid sequence of the protein encoded by the nucleotide sequence is SEQ ID No. 2.
本申请的第五种实施方式提供了NDV-VII株的全基因组，其核苷酸序列为SEQ ID No.3。The fifth embodiment of the present application provides the entire genome of the NDV-VII strain, and its nucleotide sequence is SEQ ID No. 3.
所述弱毒株经过连续20代鸡胚传代，其MDT大于120h；F基因裂解位点氨基酸序列无突变。The attenuated strain has passed through 20 consecutive generations of chicken embryos, and its MDT is greater than 120h; there is no mutation in the amino acid sequence of the cleavage site of the F gene.
本申请中的NDV-VII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(MDT)为138h，1日龄雏鸡脑内接种致病指数(ICPI)为0.16，6周龄鸡翅静脉接种致病指数(IVPI)为0。所述毒株具有稳定遗传性，在鸡胚上连续传代20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 NDV-VII strain in this application was obtained by natural isolation. The average lethal time (MDT) of this strain to chicken embryos was 138h, and the intracerebral inoculation pathogenicity index (ICPI) of 1-day-old chicks was 0.16. The IVPI of 6-week-old chicken wings intravenously was 0. The strain has stable heredity and has been successively passaged on chicken embryos for 20 generations, and its F gene does not have mutations with enhanced pathogenicity. The comparison test of the immunoprotective efficacy of the traditional vaccine strain LaSota strain proved that the antibody production level of the live vaccine prepared by this strain was higher than that of the control group. The post-immunization challenge experiment proved that the strain can provide complete protection to chickens.
附图说明Description of the drawings
图1：NDV-VII株F片段PCR鉴定结果；Figure 1: PCR identification result of F fragment of NDV-VII strain;
图2：NDV-VII株全基因组9个片段PCR扩增电泳图；Figure 2: PCR amplification electrophoresis diagram of 9 fragments of the whole genome of NDV-VII strain;
图3：基于F基因构建的***进化发育树。Figure 3: Phylogenetic development tree constructed based on F gene.
具体实施方式Detailed ways
以下结合具体实施例对本申请的技术方案进行详实的阐述，然而应当理解，在没有进一步叙述的情况下，一个实施例的实现也可以采用本领域技术人员能够想到的其他方式或手段来实现，例如且不限于实验参数的变动，实验试剂的替换等。本申请中疫苗的制备方法可以采用疫苗制备领域中常用的方法，而不仅限于本申请实施例的具体记载，本领域的普通技术人员可以其它常规方法来实现本申请。The technical solutions of the present application will be described in detail below in conjunction with specific embodiments. However, it should be understood that, without further description, the implementation of an embodiment can also be achieved by other ways or means that those skilled in the art can think of, such as It is not limited to changes in experimental parameters, replacement of experimental reagents, etc. The preparation method of the vaccine in this application can adopt methods commonly used in the field of vaccine preparation, and is not limited to the specific description of the examples of this application. Those of ordinary skill in the art can implement this application by other conventional methods.
本申请中所涉及的生物材料：LaSota活疫苗和基因VII型新城疫病毒强毒株JS02/06株由青岛易邦生物工程有限公司提供；H5、H7、H9亚型AI及ND标准阳性血清均购自中国兽医药品监察所。The biological materials involved in this application: LaSota live vaccine and genotype VII Newcastle disease virus virulent strain JS02/06 were provided by Qingdao Yibang Bioengineering Co., Ltd.; H5, H7, H9 subtype AI and ND standard positive sera were all provided Purchased from China Veterinary Drug Inspection Institute.
实施例1：NDV弱毒株的筛选及MDT、ICPI、IVPI的测定Example 1: Screening of attenuated strains of NDV and determination of MDT, ICPI, IVPI
2019年4月份从山东某地区临床送检鸡中分离到一株疑似NDV毒株，命名为NDV-VII株。接种鸡胚收取尿囊液，测定HA效价。连续传代20代，HA效价测定结果显示，该株病毒鸡胚尿囊液的HA效价在鸡胚中传代4代后稳定在9log2。收获的尿囊液进行微量血凝抑制试验(HI)，结果表明，尿囊液样品只与ND标准阳性血清反应，与H5、H7、H9阳性血清反应阴性。In April 2019, a suspected NDV strain was isolated from chickens submitted for clinical inspection in a certain area of Shandong, and was named NDV-VII strain. Allantoic fluid was collected by inoculating chicken embryos, and HA titer was determined. After 20 consecutive passages, the HA titer determination results showed that the HA titer of the chicken embryo allantoic fluid of this strain of virus stabilized at 9 log2 after 4 generations in the chicken embryo. The harvested allantoic fluid was subjected to a micro-hemagglutination inhibition test (HI). The results showed that the allantoic fluid sample only reacted with ND standard positive sera and negatively reacted with H5, H7, and H9 positive sera.
更具体地，2019年4月份从山东某养殖场送来待检测健康鸡的组织样品，取鸡的 气管、肝脏、心脏、肠道等组织，加入pH 7.2的四抗PBS(Penicilin 10000IU/mL,Streptomycin 10mg/mL,Geamicin 250μg/mL,Kanamycin 250μg/mL)进行研磨。8000r/min离心10min，取上清0.2mL接种于9-10日龄SPF鸡胚，24h内死亡的鸡胚弃掉，96h后收取鸡胚的尿囊液，测定HA效价。有效价的尿囊液倍比稀释后接种鸡胚成纤维细胞(CEF)进行蚀斑克隆纯化3次；再次接种鸡胚，96h后收取鸡胚尿囊液，测定HA效价，结果显示，该株病毒纯化后在鸡胚中传代4代，尿囊液的HA效价即可稳定在9log2；得到毒株并对其进行保藏。收获的尿囊液进行微量血凝抑制试验(HI)，结果表明，尿囊液样品只与ND标准阳性血清反应，与H5、H7、H9阳性血清反应阴性。More specifically, tissue samples of healthy chickens to be tested were sent from a farm in Shandong in April 2019. The trachea, liver, heart, intestine and other tissues of the chicken were taken, and the four antibodies PBS (Penicilin 10000IU/mL, pH 7.2) were added. Streptomycin 10mg/mL, Geamicin 250μg/mL, Kanamycin 250μg/mL) for grinding. Centrifuge at 8000r/min for 10min, take 0.2mL of supernatant and inoculate it in 9-10 day-old SPF chicken embryos, discard the chicken embryos that died within 24h, collect the allantoic fluid of the chicken embryos after 96h, and determine the HA titer. The effective value of the allantoic fluid was diluted twice and then inoculated with chicken embryo fibroblasts (CEF) for plaque cloning and purification three times; the chicken embryo was again inoculated, and the chicken embryo allantoic fluid was collected 96 hours later, and the HA titer was measured. After the virus strain is purified, it is passaged in chicken embryos for 4 generations, and the HA titer of allantoic fluid can be stabilized at 9log2; the virus strain is obtained and stored. The harvested allantoic fluid was subjected to a micro-hemagglutination inhibition test (HI). The results showed that the allantoic fluid sample only reacted with ND standard positive sera and negatively reacted with H5, H7, and H9 positive sera.
本申请中，MDT全称为Mean death time，ICPI全称为Intracephalic pathogenicity index，IVPI全称为Intravenous pathogenicity index。In this application, MDT is called Mean death time, ICPI is called Intracephalic pathogenicity index, and IVPI is called Intravenous pathogenicity index.
MDT是指最小致死量所能使鸡胚死亡的平均时间，其中，MDT低于60h为NDV强毒株，MDT值在60h-90h之间为中等毒力毒株，MDT大于90h为弱毒株。用无菌生理盐水将新鲜收获的含毒尿囊液作10倍系列稀释，取10 -6、10 -7、10 -8和10 -9共4个稀释度，分别接种10日龄SPF鸡胚，每个稀释度接种5枚，每胚尿囊腔内注射0.1mL。连续观察7日，每天上午和下午各照胚一次，记录每一鸡胚的死亡时间，连续观察7日(结果见表1)。MDT计算公式为： MDT refers to the average time for the death of chicken embryos due to the minimum lethal dose. Among them, MDT less than 60h is a virulent strain of NDV, MDT value between 60h and 90h is a moderately virulent strain, and MDT greater than 90h is a weakly virulent strain. Use sterile normal saline to dilute the freshly harvested toxic allantoic fluid in a 10-fold series, and take 4 dilutions of 10 -6 , 10 -7 , 10 -8 and 10 -9 to inoculate 10-day-old SPF chicken embryos respectively , Inoculate 5 in each dilution, and inject 0.1 mL into the allantoic cavity of each embryo. Observe for 7 consecutive days, take the embryos once in the morning and afternoon each day, record the death time of each embryo, and observe for 7 consecutive days (see Table 1 for the results). The MDT calculation formula is:
Figure PCTCN2020088921-appb-000001
Figure PCTCN2020088921-appb-000001
1日龄SPF雏鸡脑内致病指数(ICPI)测定试验：取1日龄SPF雏鸡10只，其中5只各脑内注射用PBS(phosphate buffer saline)10倍稀释的新鲜含毒尿囊液0.05mL；剩余5只，以同样方法注射相同剂量的无菌PBS作为对照。接种后，连续观察8日，每日在相应接种的时间观察，记录雏鸡的情况，分正常(活动灵活，行动 无共济失调现象)、发病(包括麻痹、卧地不起，但不包括只表现迟钝的鸡)和死亡，结果见表2。1-day-old SPF chicks intracerebral pathogenicity index (ICPI) test: Take 10 1-day-old SPF chicks, 5 of which were injected into the brain with fresh toxic allantoic fluid diluted 10 times with PBS (phosphate buffer saline) 0.05 mL; the remaining 5 animals were injected with the same dose of sterile PBS in the same way as a control. After vaccination, observe for 8 consecutive days, observe the corresponding vaccination time every day, record the condition of the chicks, divide into normal (flexible activities, no ataxia), disease (including paralysis, lying on the ground, but not including chickens). Chickens with slow performance) and death. The results are shown in Table 2.
Figure PCTCN2020088921-appb-000002
Figure PCTCN2020088921-appb-000002
6周龄SPF雏鸡静脉接种致病指数(IVPI)测定试验：采用PBS 10倍稀释新鲜感染尿囊液，取6周龄SPF小鸡10只，其中5只各翅静脉接种0.1mL；剩余5只，以同样方法注射相同剂量的无菌PBS作为对照。接种后，连续观察10日，每日在相应接种的时间观察，记录雏鸡的情况，分正常(活动灵活，行动无共济失调现象)、发病(包括瘫痪、卧地不起，但不包括只表现迟钝的鸡)和死亡，结果见表3。6-week-old SPF chicks intravenous inoculation pathogenicity index (IVPI) test: Dilute the freshly infected allantoic fluid with PBS 10 times, and take 10 6-week-old SPF chicks, 5 of which were intravenously inoculated with 0.1 mL of each wing; the remaining 5 , Inject the same dose of sterile PBS in the same way as a control. After vaccination, observe continuously for 10 days. Observe at the corresponding vaccination time every day. Record the condition of the chicks. They are divided into normal (flexible activities, no ataxia), disease (including paralysis, lying on the ground, but not including chicks). Chickens that behaved sluggishly) and died. The results are shown in Table 3.
Figure PCTCN2020088921-appb-000003
Figure PCTCN2020088921-appb-000003
表1：NDV-VII株MDT的测定Table 1: MDT determination of NDV-VII strain
Figure PCTCN2020088921-appb-000004
Figure PCTCN2020088921-appb-000004
表2：NDV-VII株ICPI的测定Table 2: Determination of ICPI of NDV-VII strain
Figure PCTCN2020088921-appb-000005
Figure PCTCN2020088921-appb-000005
表3：NDV-VII株IVPI测定Table 3: IVPI determination of NDV-VII strain
Figure PCTCN2020088921-appb-000006
Figure PCTCN2020088921-appb-000006
Figure PCTCN2020088921-appb-000007
Figure PCTCN2020088921-appb-000007
表1～表3显示，NDV-VII株的MDT为138h，ICPI为0.16，IVPI为0，符合NDV弱毒特征，证明本次分离到的NDV-VII株为弱毒株。Tables 1 to 3 show that the MDT of the NDV-VII strain is 138h, the ICPI is 0.16, and the IVPI is 0, which conforms to the attenuated characteristics of NDV, which proves that the NDV-VII strain isolated this time is an attenuated strain.
对本次筛选获得的基因VII型新城疫病毒弱毒株NDV-VII株，根据《国际承认用于专利程序的微生物保藏布达佩斯条约》，已于2019年10月15日保藏在位于武汉大学的中国典型培养物保藏中心，其保藏号为CCTCC V201968。The NDV-VII strain of the attenuated genotype VII Newcastle disease virus obtained in this screening has been deposited at Wuhan University on October 15, 2019 in accordance with the Budapest Treaty on the International Recognition of the Deposit of Microorganisms Used in Patent Procedures. The Culture Collection, its deposit number is CCTCC V201968.
实施例2：NDV-Ⅶ株F基因鉴定及进化树分析Example 2: Identification of F gene of NDV-Ⅶ strain and phylogenetic tree analysis
根据NCBI公布的NDV的F基因序列设计引物，上游引物：NDV F-F：ATGGGCTCCAAACCTTCTACCAG；下游引物：NDV F-R：AAACTGCTGCATCTTCCCAACCG，进行NDV-VII株的F片段扩增鉴定，目的片段约550bp。取鸡胚传代收获的尿囊液200uL按常规方法提取RNA，反转录，F基因片段的核苷酸序列为SEQ ID No.1，该核苷酸序列编码蛋白的氨基酸序列为SEQ ID No.2。核酸电泳后(结果如图1所示)，切取阳性条带，回收，连接T载体测序。取F基因47nt-420nt之间的序列绘制***进化发育树，结果如图3所示。The primers were designed according to the F gene sequence of NDV published by NCBI. Upstream primer: NDV F-F: ATGGGCTCCAAACCTTCTACCAG; downstream primer: NDV F-R: AAACTGCTGCATCTTCCCAACCG. The F fragment of the NDV-VII strain was amplified and identified, and the target fragment was about 550 bp. Take 200uL of allantoic fluid harvested from chicken embryo passages to extract RNA according to conventional methods, reverse transcription, the nucleotide sequence of the F gene fragment is SEQ ID No. 1, and the amino acid sequence of the nucleotide sequence encoding the protein is SEQ ID No. 2. After nucleic acid electrophoresis (the result is shown in Figure 1), the positive bands were cut out, recovered, and connected to the T vector for sequencing. Take the sequence between 47nt-420nt of the F gene to draw a phylogenetic development tree, and the result is shown in Figure 3.
结果显示，所分离的NDV-VII株属于ClassⅡ亚群，基因Ⅶ型新城疫毒株，与近年中国多地流行株处于同一进化分支，同属于Ⅶd亚型。The results showed that the isolated NDV-VII strain belonged to the Class II subgroup, and the gene type VII Newcastle disease strain was in the same evolutionary branch as the strains circulating in many places in China in recent years, and belonged to the same subtype VIId.
实施例3：NDV-VII株全基因组序列的测定Example 3: Determination of the whole genome sequence of NDV-VII strain
根据NCBI公布的NDV基因序列，设计9对引物，相邻扩增片段之间有部分核酸序列相互重叠。引物由上海生工生物工程有限公司合成，序列见表4。According to the NDV gene sequence published by NCBI, 9 pairs of primers were designed, and some nucleic acid sequences between adjacent amplified fragments overlapped with each other. The primers were synthesized by Shanghai Shenggong Biological Engineering Co., Ltd., and the sequence is shown in Table 4.
表4：扩增NDV-VII株全基因组的引物序列Table 4: Sequences of primers used to amplify the entire genome of the NDV-VII strain
Figure PCTCN2020088921-appb-000008
Figure PCTCN2020088921-appb-000008
常规柱提法提取病毒RNA，随机引物进行反转录，用上述引物进行PCR扩增，扩增片段电泳扩增结果见图2。将目的片段切胶回收后连接PMD 19-T载体，转化、测序，序列在NCBI网站比对，用Lasergene软件拼接，获得NDV-VII株全基因序列(SEQ ID No.3)。Conventional column extraction method is used to extract viral RNA, random primers are used for reverse transcription, and the above primers are used for PCR amplification. The results of electrophoresis amplification of the amplified fragments are shown in Figure 2. The target fragment was cut and recovered and connected to the PMD 19-T vector, transformed and sequenced. The sequence was compared on the NCBI website and assembled with Lasergene software to obtain the full gene sequence of the NDV-VII strain (SEQ ID No. 3).
F基因全长1662bp，裂解位点处氨基酸序列为 112G-R-Q-G-R-L 117，符合弱毒株裂解位点特征。 The F gene has a full length of 1662 bp, and the amino acid sequence at the cleavage site is 112 GRQGRL 117 , which conforms to the characteristics of the attenuated strain cleavage site.
根据序列SEQ ID No.3可知，NDV-VII株基因组全长15186bp，与常规分离的基 因Ⅶ型NDV基因组全长(15192bp)不一致，推测NDV-VII株在临床长期的流行过程中发生过重组，并最终稳定遗传。该病毒F基因裂解位点处氨基酸特征与毒力有密切的关系，根据全基因组序列可知，该毒株F基因裂解位点处氨基酸序列为 112G-R-Q-G-R-L 117，符合弱毒株裂解位点的普遍特征，说明该分离株NDV-VII具有弱毒的特征。 According to the sequence SEQ ID No.3, the NDV-VII strain has a full-length genome of 15186bp, which is inconsistent with the conventionally isolated gene type VII NDV genome (15192bp). It is speculated that the NDV-VII strain has undergone recombination during the long-term clinical epidemic. And finally stable inheritance. The amino acid characteristics at the cleavage site of the F gene of the virus are closely related to virulence. According to the whole genome sequence, the amino acid sequence at the cleavage site of the F gene of this strain is 112 GRQGRL 117 , which is consistent with the general characteristics of the attenuated strain’s cleavage site. This indicates that the isolate NDV-VII has the characteristics of attenuating toxicity.
实施例4：NDV-VII株鸡胚传代稳定性试验Example 4: Passage Stability Test of Chicken Embryo of NDV-VII Strain
将NDV-VII株通过鸡胚进行连续传代试验，每隔5代测定病毒MDT及ICPI，并对F基因进行测序、分析裂解位点氨基酸序列，传至20代。结果发现，经过连续20代鸡胚传代，该毒株MDT始终大于120h，F基因裂解位点氨基酸序列并无突变，证明该毒株虽然可能是自然重组毒株，但遗传性能稳定，适合作为疫苗株候选株(表5)。The NDV-VII strain was passed through chicken embryos for serial passage experiments, virus MDT and ICPI were measured every 5 generations, and the F gene was sequenced, and the amino acid sequence of the cleavage site was analyzed to pass to 20 generations. The results showed that after 20 consecutive generations of chicken embryos, the MDT of the strain was always greater than 120h, and there was no mutation in the amino acid sequence at the cleavage site of the F gene, which proved that although the strain may be a naturally recombined strain, its genetic performance is stable and suitable as a vaccine Candidate strains (Table 5).
表5：NDV-VII株MDT及F基因裂解位点Table 5: MDT and F gene cleavage sites of NDV-VII strain
Figure PCTCN2020088921-appb-000009
Figure PCTCN2020088921-appb-000009
实施例5：NDV-VII株保存期稳定性试验Example 5: Storage period stability test of NDV-VII strain
将收获的传代20代NDV-VII株新鲜病毒尿囊液(EID 50为10 9.17/0.1mL)等量分成若干份置于-80℃保存，分别于保存期内1天、7天、14天、28天、2月、4月、6月取出，4℃溶化后测定HA效价，并同时接种9-11日龄SPF鸡胚，0.1mL/枚，37℃培养至120h，收取尿囊液测定HA效价，检验病毒的保存稳定性。 Divide the harvested fresh virus allantoic fluid (EID 50 of 10 9.17/ 0.1 mL) of the 20-generation NDV-VII strain harvested into several equal portions and store them at -80°C for 1 day, 7 days, and 14 days during the storage period. , 28 days, February, April, June, take out, 4 ℃ thawed, determine HA titer, and simultaneously inoculate 9-11 day old SPF chicken embryos, 0.1mL/piece, 37 ℃ culture to 120h, collect allantoic fluid Determine the HA titer and check the storage stability of the virus.
结果显示，病毒尿囊液保存至-80℃条件下6个月，HA并无显著性下降，第21代收获的尿囊液HA无明显下降，说明该代次病毒在-80℃条件下较为稳定，至少能够保存6个月(表6)。The results showed that the virus allantoic fluid was stored at -80℃ for 6 months, the HA did not decrease significantly, and the HA of the allantoic fluid harvested at the 21st generation did not decrease significantly, indicating that the virus of this generation was relatively low at -80℃. It is stable and can be stored for at least 6 months (Table 6).
表6：NDV-VII株保存期内HA测定结果Table 6: HA determination results of NDV-VII strain during storage period
Figure PCTCN2020088921-appb-000010
Figure PCTCN2020088921-appb-000010
实施例6：NDV-VII株活疫苗的制备及安全性试验、无菌检验Example 6: Preparation, safety test and sterility test of NDV-VII strain live vaccine
用5％蔗糖脱脂乳1:1加入至稀释的尿囊液中，定量稀释后的病毒含量为10 7.0EID 50/0.1mL，充分混匀后冻干得到冻干活疫苗，以保存疫苗；需要使用时采用PBS溶液稀释即可。制备的活疫苗按2010年版《中国兽药典》附录进行无菌检验，结果符合标准，无细菌污染。 Add 5% sucrose skimmed milk to the diluted allantoic fluid 1:1, the virus content after quantitative dilution is 10 7.0 EID 50 /0.1mL, mix well and freeze-dry to obtain the freeze-dried live vaccine to preserve the vaccine; Use PBS solution to dilute it when in use. The prepared live vaccines were tested for sterility in accordance with the 2010 edition of the "Chinese Veterinary Pharmacopoeia" appendix, and the results met the standards without bacterial contamination.
用1～3日龄SPF鸡10只，分为实验组5只，PBS对照组5只；其中实验组每只免疫10 7.0EID 50的病毒液，对照组免疫相同体积的无菌PBS，免疫方式为滴鼻点眼。在相同的条件下饲养，连续观察14日，记录试验鸡采食、饮水及临床表现。结果显示，实验组免疫后鸡只与对照组一样，没有出现精神沉郁、食欲不振、嗜睡麻痹及全身不良反应，鸡只健康，证明制备的NDV-VII活疫苗安全。 10 SPF chickens aged 1 to 3 days were divided into 5 experimental groups and 5 PBS control groups; each of the experimental group was immunized with 10 7.0 EID 50 virus solution, and the control group was immunized with the same volume of sterile PBS. Drop the eyes for the nose. They were raised under the same conditions and observed for 14 consecutive days, and the food intake, drinking water and clinical manifestations of the test chickens were recorded. The results showed that the chickens in the experimental group were the same as the control group after immunization. They did not suffer from depression, loss of appetite, lethargy, paralysis and systemic adverse reactions. The chickens were healthy, which proved the safety of the prepared NDV-VII live vaccine.
实施例7：NDV-VII株活疫苗免疫效果评价Example 7: Evaluation of Immunity Effect of NDV-VII Strain Live Vaccine
用21日龄SPF鸡30只，随机分为3组，每组10只，其中，第一组为NDV-VII株冻干活疫苗组，第二组为LaSota株活疫苗组，以及第三组为PBS对照组，滴鼻点眼免疫，0.1mL/只，免疫后每组隔离饲养，分别于免后14天、21天、28天采血，测定抗体。28天采血后，用基因VII型强毒株JS 02/06株攻毒，攻毒方式为滴鼻点眼，剂量为10 6EID 50/只，隔离器饲养，观察14天，每天记录鸡的发病和死亡情况；攻毒后第3天及第5天，分别采集各免疫组鸡的喉拭子和泄殖腔拭子接胚，检测排毒情况。 30 SPF chickens aged 21 days were randomly divided into 3 groups, each with 10 chickens. Among them, the first group is the NDV-VII strain freeze-dried live vaccine group, the second group is the LaSota strain live vaccine group, and the third group For the PBS control group, immunized with nasal drops and eye drops, 0.1 mL/moist. After immunization, each group was reared in isolation, and blood was collected at 14 days, 21 days, and 28 days after immunization to determine antibodies. After 28 days of blood collection, challenge with genotype VII virulent strain JS 02/06. The method of challenge is nasal drops and eye drops at a dose of 10 6 EID 50 per bird. The animals are raised in an isolator. Observe for 14 days and record the incidence of chickens every day. And death; on the 3rd and 5th day after the challenge, the throat swabs and cloacal swabs of the immunized chickens were collected and embryos were collected to detect the detoxification status.
结果显示，第一组和第二组免疫14天即可产生较高水平抗体，21天、28天抗体水平升高，结果如表7所示。免疫攻毒后，第一组和第二组均没有鸡只死亡，第三组鸡全部死亡。排毒试验显示，第一组没有出现排毒，而第二组在第五天在喉气管及泄 殖腔均有排毒，见表8和表9。The results showed that the first group and the second group could produce higher levels of antibodies within 14 days of immunization, and the antibody levels increased on the 21st and 28th days. The results are shown in Table 7. After the immunization challenge, none of the chickens in the first and second groups died, and all the chickens in the third group died. The detoxification test showed that the first group did not detox, while the second group had detoxification in the larynx, trachea and cloaca on the fifth day, see Table 8 and Table 9.
表7：免疫后抗体效价测定表Table 7: Antibody titer determination table after immunization
Figure PCTCN2020088921-appb-000011
Figure PCTCN2020088921-appb-000011
表8：免疫效力实验结果表Table 8: Immune efficacy test result table
Figure PCTCN2020088921-appb-000012
Figure PCTCN2020088921-appb-000012
表9:攻毒后第3天、5天病毒分离结果Table 9: Virus isolation results on the 3rd and 5th days after challenge
Figure PCTCN2020088921-appb-000013
Figure PCTCN2020088921-appb-000013
综上所述，本申请所构建的NDV-VII株，具有良好的免疫原性，做成活疫苗免疫动物发现，该疫苗对鸡只安全，能够诱导产生较高的中和抗体，并且能够保护当前流行的基因VII型强毒株的攻击，产生很好的保护效果，有广阔的应用前景。In summary, the NDV-VII strain constructed in this application has good immunogenicity. It was found that the vaccine is safe for chickens, can induce the production of higher neutralizing antibodies, and can protect animals. The currently popular Genotype VII virulent strain has a very good protective effect and has broad application prospects.
所述的实施方式仅仅是对本申请的优选实施方式进行描述，并非对本申请的范围进行限定，在不脱离本申请设计精神的前提下，本领域普通技术人员对本申请的技术方案作出的各种变形和改进，均应落入本申请权利要求书确定的保护范围内。The described implementations are merely descriptions of the preferred implementations of the application, and are not intended to limit the scope of the application. Without departing from the design spirit of the application, various modifications made by those of ordinary skill in the art to the technical solutions of the application All improvements and improvements shall fall within the scope of protection determined by the claims of this application.
Figure PCTCN2020088921-appb-000014
Figure PCTCN2020088921-appb-000014
Figure PCTCN2020088921-appb-000015
Figure PCTCN2020088921-appb-000015
Figure PCTCN2020088921-appb-000016
Figure PCTCN2020088921-appb-000016
Figure PCTCN2020088921-appb-000017
Figure PCTCN2020088921-appb-000017
Figure PCTCN2020088921-appb-000018
Figure PCTCN2020088921-appb-000018
Figure PCTCN2020088921-appb-000019
Figure PCTCN2020088921-appb-000019
Figure PCTCN2020088921-appb-000020
Figure PCTCN2020088921-appb-000020
Figure PCTCN2020088921-appb-000021
Figure PCTCN2020088921-appb-000021
Figure PCTCN2020088921-appb-000022
Figure PCTCN2020088921-appb-000022
Figure PCTCN2020088921-appb-000023
Figure PCTCN2020088921-appb-000023
Figure PCTCN2020088921-appb-000024
Figure PCTCN2020088921-appb-000024

Claims (10)

  1. 一种基因VII型新城疫病毒弱毒株，其中，所述的弱毒株的保藏编号为CCTCCV201968。A genotype VII Newcastle disease virus attenuated strain, wherein the attenuated strain has a deposit number of CCTCCV201968.
  2. 根据权利要求1所述的弱毒株，其中，所述弱毒株的F基因的核苷酸序列为SEQ ID No.1。The attenuated strain according to claim 1, wherein the nucleotide sequence of the F gene of the attenuated strain is SEQ ID No. 1.
  3. 根据权利要求2所述的弱毒株，其中，所述F基因编码蛋白的氨基酸序列为SEQ ID No.2。The attenuated strain according to claim 2, wherein the amino acid sequence of the protein encoded by the F gene is SEQ ID No.2.
  4. 根据权利要求1所述的弱毒株，其中，所述弱毒株的全基因组的核苷酸序列为SEQ ID No.3。The attenuated strain according to claim 1, wherein the nucleotide sequence of the entire genome of the attenuated strain is SEQ ID No. 3.
  5. 根据权利要求1-4任一项所述的弱毒株，其中，经过连续20代鸡胚传代，所述弱毒株的MDT大于120h。The attenuated strain according to any one of claims 1 to 4, wherein after 20 consecutive generations of chicken embryos, the MDT of the attenuated strain is greater than 120h.
  6. 根据权利要求5所述的弱毒株，其中，所述弱毒株的F基因裂解位点氨基酸序列无突变。The attenuated strain according to claim 5, wherein there is no mutation in the amino acid sequence of the cleavage site of the F gene of the attenuated strain.
  7. 一种权利要求1所述的弱毒株在制备疫苗中的应用。An application of the attenuated strain of claim 1 in preparing a vaccine.
  8. 根据权利要求7所述的应用，其中，所述的疫苗为活毒疫苗。The application according to claim 7, wherein the vaccine is a live vaccine.
  9. 一种疫苗，其中，所述的疫苗中的抗原包含有权利要求1所述的弱毒株。A vaccine, wherein the antigen in the vaccine comprises the attenuated strain of claim 1.
  10. 根据权利要求9所述的疫苗，其中，所述疫苗为活疫苗。The vaccine according to claim 9, wherein the vaccine is a live vaccine.
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114292823A (en) * 2021-12-17 2022-04-08 河南农业大学 Recombinant LaSota vaccine strain carrying genes VII type Newcastle disease virus F and HN genes and construction method and application thereof
CN116004552A (en) * 2023-02-20 2023-04-25 北京市农林科学院 Pigeon paramyxovirus type I recombinant attenuated vaccine strain rGX-mF and application and vaccine thereof

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110846287B (en) * 2019-11-27 2022-09-09 青岛易邦生物工程有限公司 Gene VII type Newcastle disease virus attenuated strain and application thereof
CN114164184B (en) * 2020-09-10 2024-02-09 青岛易邦生物工程有限公司 Newcastle disease virus gene VI vaccine strain and application thereof
CN112877361A (en) * 2021-02-08 2021-06-01 青岛海华生物医药技术有限公司 Recombinant Newcastle disease virus weak vaccine strain and construction method thereof

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101182494A (en) * 2007-09-05 2008-05-21 扬州大学 Gene VII type new castle disease virus weakening strain A-NDV-VII and construction method thereof
CN105543180A (en) * 2016-01-04 2016-05-04 山东省农业科学院畜牧兽医研究所 Isolation, identification and purification method and application of gene VII type Newcastle disease virus strain
CN107164335A (en) * 2017-05-27 2017-09-15 山东信得科技股份有限公司 A kind of genotype VII Newcastle disease virus strain
CN107213460A (en) * 2017-05-27 2017-09-29 山东信得科技股份有限公司 A kind of genotype VII newcastle disease vaccine
CN107281479A (en) * 2016-03-31 2017-10-24 普莱柯生物工程股份有限公司 A kind of genotype Ⅶ NDV low virulent strain, vaccine combination and its application
CN110846287A (en) * 2019-11-27 2020-02-28 青岛易邦生物工程有限公司 Gene VII type Newcastle disease virus attenuated strain and application thereof

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104031888B (en) * 2014-03-26 2016-04-06 东北林业大学 Newcastle disease virus low virulent strain, inactivated vaccine and application thereof
CN105985966A (en) * 2015-03-06 2016-10-05 普莱柯生物工程股份有限公司 Gene VII-type newcastle disease virus strain, vaccine composition thereof and preparing method and application of vaccine composition

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101182494A (en) * 2007-09-05 2008-05-21 扬州大学 Gene VII type new castle disease virus weakening strain A-NDV-VII and construction method thereof
CN105543180A (en) * 2016-01-04 2016-05-04 山东省农业科学院畜牧兽医研究所 Isolation, identification and purification method and application of gene VII type Newcastle disease virus strain
CN107281479A (en) * 2016-03-31 2017-10-24 普莱柯生物工程股份有限公司 A kind of genotype Ⅶ NDV low virulent strain, vaccine combination and its application
CN107164335A (en) * 2017-05-27 2017-09-15 山东信得科技股份有限公司 A kind of genotype VII Newcastle disease virus strain
CN107213460A (en) * 2017-05-27 2017-09-29 山东信得科技股份有限公司 A kind of genotype VII newcastle disease vaccine
CN110846287A (en) * 2019-11-27 2020-02-28 青岛易邦生物工程有限公司 Gene VII type Newcastle disease virus attenuated strain and application thereof

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114292823A (en) * 2021-12-17 2022-04-08 河南农业大学 Recombinant LaSota vaccine strain carrying genes VII type Newcastle disease virus F and HN genes and construction method and application thereof
CN116004552A (en) * 2023-02-20 2023-04-25 北京市农林科学院 Pigeon paramyxovirus type I recombinant attenuated vaccine strain rGX-mF and application and vaccine thereof
CN116004552B (en) * 2023-02-20 2023-08-25 北京市农林科学院 Pigeon paramyxovirus type I recombinant attenuated vaccine strain rGX-mF and application and vaccine thereof

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