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Separation, identification and application of avian H9N2 subtype avian influenza virus strain

An avian influenza virus and avian influenza technology, which is applied in the field of animal virology, can solve the problems of egg production decline of laying hens, serious respiratory diseases secondary to flocks, and impact on poultry production performance, etc., and achieve excellent protection effect and protection against attack. Effect

Active Publication Date: 2020-04-24
BEIJING HUAXIA XINGYANG BIOLOGICAL SCI & TECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, AIV is widely present in poultry flocks all over the world. The incidence of H9N2 subtype accounts for more than 90% of the total incidence of avian influenza. Although the fatality rate is less than 30%, it can lead to respiratory symptoms and a drop in egg production of laying hens. , and make chickens prone to secondary severe respiratory diseases, affecting poultry production performance

Method used

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  • Separation, identification and application of avian H9N2 subtype avian influenza virus strain
  • Separation, identification and application of avian H9N2 subtype avian influenza virus strain
  • Separation, identification and application of avian H9N2 subtype avian influenza virus strain

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1 Virus isolation and identification

[0031] Take the throat cavity and cloaca swabs of sick chickens, treat them in PBS buffer containing penicillin and streptomycin (600IU / ml) for 2 hours, fully twist and squeeze, retain the liquid part and centrifuge at 8000rpm for 10min, and take the supernatant 0.22um filtration treatment, inoculation of 0.2ml / piece of 9-11-day-old SPF chicken embryos, collecting allantoic fluid of dead embryos after 24 hours, and identifying its purity by RT-PCR and hemagglutination tests. The allantoic fluid of mixed infection samples was neutralized by adding specific positive serum at 1:1, combined with chicken embryo terminal dilution method to continue passage. Until two consecutive generations of allantoic fluid were tested, the hemagglutination value of H9N2 was not lower than 5Log2, and the hemagglutination value of other subtypes of AIV and NDV was not higher than 3Log2; at the same time, RT-PCR was positive for H9, and all other...

Embodiment 2

[0035] Embodiment 2 virus HA, NA gene amplification and sequence determination

[0036] 1. Primer Synthesis

[0037] According to the sequence published by Genbank, HA and NA-specific full-length primers were designed with Primer5 software, as shown in Table 2.

[0038] Table 2 Identification and sequencing primers

[0039]

[0040] 2. Target gene amplification and sequence determination

[0041] The present invention uses purified allantoic fluid that can stably proliferate in SPF chicken embryos as a nucleic acid extraction sample, and operates according to the instructions of the RNApure ultra-pure total RNA rapid extraction kit. The extracted RNA was amplified by RT-PCR according to the method recommended by EasyScript One-StepEnzyme Mix. After 1% agarose gel electrophoresis proved that the target band was amplified, the RT-PCR amplification product was sent to Beijing Sangon Biotechnology Co., Ltd. for sequencing. The obtained HA gene amino acid sequence is shown i...

Embodiment 3

[0042] Embodiment 3 virus HA antigenicity analysis

[0043] 1. Single Factor Serum Preparation

[0044]A total of 8 H9N2 epidemic strains isolated in 2018, including the AIV-BZ strain involved in the present invention, were mixed according to the ratio of inactivated venom: 605 adjuvant 4:6 to prepare inactivated vaccines, and immunized 21-day-old SPF chickens, Raised in an isolator for 14 days, blood was collected to separate serum, and single-factor serum was prepared.

[0045] 2. Crossover HI test

[0046] Prepare 4 units of virus solution of the above 8 strains of virus respectively, and conduct cross-HI test with the above-mentioned 8 kinds of single-factor sera to detect the cross-reactivity of HA antigen. Repeat three times and take the average value to determine the correlation R between the above-mentioned H9N2 virus HA antigens.

[0047] The criteria for judging antigenic relevance are as follows:

[0048] The correlation between antigens R=√(R1xR2),

[0049] R:...

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Abstract

The invention relates to the field of animal virology, and provides separation, identification and application of an avian H9N2 subtype avian influenza virus strain. The H9N2 subtype avian influenza virus strain is preserved in China General Microbiological Culture Collection Center on July 02, 2019 with the preservation number of CGMCC No. 18172. The antigen variation condition of the separated strain is explained from the molecular level; the virus strain is inactivated with formaldehyde, and then is processed together with a 605 adjuvant to prepare an inactivated vaccine, 21-day-old SPF chickens are immunized, the immune effect is evaluated through a serological method and an immune challenge method, it is proved that the strain is good in protection effect, and the strain can be used as an avian influenza H9N2 subtype vaccine candidate strain. The invention provides an idea for bird flu prevention and control, provides technical teaching for bird flu candidate strain screening, andhas great public health significance.

Description

technical field [0001] The invention relates to the field of animal virology. The invention provides research on the biological characteristics of an avian H9N2 subtype avian influenza virus strain and its application in vaccine development. [0002] technical background [0003] Avian influenza virus (AIV) belongs to the Orthomyxoviridae Influenza A virus genus. The nucleic acid is a single-stranded negative-sense RNA with a total length of about 13.6 kb. It contains 8 segments and can encode 11 proteins. Due to the characteristic that the HA segment and the NA segment in the single-stranded negative-sense RNA are most likely to mutate, when different viruses invade the same host, gene rearrangement between different viruses is very likely to occur, increasing the chance of virus mutation, and may even produce new viruses. Influenza viruses can change the range of virus host invasion and virus virulence. According to the degree of pathogenicity of AIV to poultry, it can be ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/00C07K14/11C12N15/44C12N15/11A61K39/145A61P31/16C12R1/93
CPCC12N7/00C07K14/005C12N15/11A61K39/12A61P31/16C12N2760/16121C12N2760/16151C12N2760/16122C12N2760/16134A61K2039/552A61K2039/5252
Inventor 贺亚奇尚川川武沛泽王顺山吴国彬王延博赵卓胡义彬商云鹏李晓亮金扩世王力王秀敏江厚生
Owner BEIJING HUAXIA XINGYANG BIOLOGICAL SCI & TECH
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