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Screening method and application of a cold-adapted strain of chicken-derived h9n2 avian influenza virus
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A bird flu virus, cold adaptation technology, applied in the field of animal virology, can solve the problems of inability to provide mucosal immune protection, inability to obtain cellular immunity, etc.
Inactive Publication Date: 2021-09-14
INST OF ANIMAL SCI & VETERINARY MEDICINE SHANDONG ACADEMY OF AGRI SCI
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[0005] At present, the vaccines used to prevent the outbreak of H9N2 avian influenza in China are mainly inactivated vaccines. Since inactivated vaccines mainly provide humoral immunity and can stimulate the body to produce antibodies, but cannot provide mucosal immune protection, effective cellular immunity cannot be obtained after chicken flocks are immunized. Therefore, many chicken farms have been immunized with the H9 vaccine, but there are still sporadic cases
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Embodiment 1
[0021] Embodiment 1: Obtaining method of H9N2 cold-adapted strain (SD165)
[0022] A / Chicken / Shandong / 165 / 2013 (H9N2) (SD 165 for short) is a three-generation purified virus that is continuously passed on 9-10-day-old SPF chicken embryos.
[0023] Dilute SD 165 10,000 times, and continuously passage on 10-day-old SPF chicken embryos, put 20 SPF chicken embryos inoculated with SD 165 in the first generation into a 35°C incubator, and collect allantoic fluid aseptically after 72 hours. Chicken embryoallantoic fluid with hemagglutination and hemagglutinationtiter higher than 24 continued to inoculate 10-day-old SPF chicken embryos, and the temperature of the incubator was lowered to 34.5°C. Follow the above procedure until the temperature of the incubator drops to 25°C, collect the allantoic fluid of chicken embryos with the highest hemagglutination, and continuously passage at 25°C for 10 generations, and finally maintain the hemagglutination titer of chicken embryos at 2 9 ....
Embodiment 2
[0024] Embodiment 2: Determination of the properties and immune effect of H9 subtype AIV
[0025] 1. Genetic evolution analysis: The full gene sequence of the virus is as shown in the instructions. The inventors referred to the published H9 subtype AIV sequence data, designed and synthesized 8 gene fragments of the AIV genome (PB2, PB1, PA, HA, RT-PCR primers for NA, NP, M, NS). Each gene fragment of H9 subtype AIV was amplified separately. The whole genome sequence of the virus shows that its HA gene belongs to the H9-4.2.5.2like lineage, which belongs to the same evolutionary branch as the domestic H9N2 epidemic strain in recent years, and can be used as one of the candidate strains of the H9N2 vaccine strain. Its specific gene evolution tree is attached figure 1 , the analysis software adopted by the inventor is DNAstar7.0.
[0026] 2. The growth characteristics of the virus in chicken embryos At 25°C, the virus can grow at high titers in chicken embryos, and the hemaggl...
Embodiment 3
[0043] Example 3: Deposit Information
[0044] Preservation time: December 21, 2017
[0045] Name of depository unit: CGMCC, General Microorganism Center of China Committee for Microorganism Culture Collection
[0046] Deposit number: CGMCC NO: 14887.
[0047] Address of Preservation Unit: Institute of Microbiology, Chinese Academy of Sciences
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Abstract
The present invention relates to the field of animal virology. The present invention provides a method for preparing a cold-adapted strain of chicken-origin H9N2 avian influenza virus (SD 165) passed down continuously at low temperature and its biological characteristics, as well as its application in biology and its preservation The number is CGMCC NO: 14887; After SD 165 is made into a vaccine, 1-week-old SPF chickens are immunized. Through the analysis of the protective effect against homologous and heterologous H9N2 wild strains of avian influenza, it is proved that SD 165 can be used as a vaccine for H9 subtype avian influenza Live vaccine backup strains. The invention provides vaccine spare strains for preventing and controlling bird flu outbreaks by using live vaccines of H9N2 subtype cold-adapted strains of bird flu, and provides a technical solution for how to screen live vaccine strains of bird flu, which has important public health significance.
Description
technical field [0001] The present invention relates to the field of animal virology, and the present invention provides a screening method for a cold-adapted strain (SD 165) of chicken-origin H9N2 avian influenza virus and its biological characteristics, as well as its biological effects, especially in the immunity of SPF chickens. application. Background technique [0002] Avian influenza virus (AIV) belongs to the genusInfluenzavirus A of the Orthomyxoviridae family, and its genome is divided into 8 segments. The total length of the nucleic acid is about 13.6kb, encoding at least 11 viral proteins. Due to the negative strand and segmented RNAvirus nature of AIV, the virus is easy to mutate and constantly breaks through the species barrier of its infected host, causing new influenza epidemics. AIV can be divided into highly pathogenic AIV (Highly PathogenicAvian Influenza virus, HPAlV) and low pathogenic AIV (Low PathogenicAvian Influenza virus, LPAIV) according to i...
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