H9N2 avian influenza virus vaccine strain and application of H9N2 avian influenza virus vaccine strain in immune protection

A technology of avian influenza virus and vaccine strains, applied in the field of animal virology, can solve problems such as chickens prone to secondary respiratory diseases, immune suppression, and affecting poultry production performance

Inactive Publication Date: 2012-04-04
INST OF ANIMAL SCI & VETERINARY MEDICINE SHANDONG ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Although the fatality rate of H9N2 subtype avian influenza is not high (generally no more than 30%), it often leads to respiratory symptoms, lower egg production of laying hens, and makes chickens prone to secondary severe respiratory diseases, affecting poultry production performance; Immunosuppression caused by infection of broiler chickens is likely to cause secondary infection and mixed infection, thus causing huge economic losses to the broiler chicken farming industry

Method used

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  • H9N2 avian influenza virus vaccine strain and application of H9N2 avian influenza virus vaccine strain in immune protection
  • H9N2 avian influenza virus vaccine strain and application of H9N2 avian influenza virus vaccine strain in immune protection
  • H9N2 avian influenza virus vaccine strain and application of H9N2 avian influenza virus vaccine strain in immune protection

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Experimental program
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Effect test

Embodiment 1

[0038]A H9N2 avian influenza virus vaccine strain, that is, a recombinant chicken-derived H9N2 avian influenza virus strain A / Chicken / Shangong / SG2 / 2009, the isolation and identification methods of the virus are as follows:

[0039] 1. Treatment of disease materials and inoculation of chicken embryo allantoic cavity

[0040] Aseptically grind the collected disease materials, collect the extract, or repeatedly freeze the nasopharyngeal or tracheal swabs in normal saline for 3 times, then centrifuge at low speed, collect the supernatant, and collect the supernatant in the SPF chicken embryos aged 9 to 11 days. Inoculate 0.2ML of virus fluid into the allantoic cavity of the human body, incubate at 35°C-37°C, collect the allantoic fluid of chicken embryos that died naturally or did not die within 24-72 hours, use hemagglutination inhibition and PCR methods to identify virus subtypes, and PCR to identify primers See Table 1-1; in the process of continuous passage, select the virus l...

Embodiment 2

[0061] experimental method:

[0062] 1. Preparation of H9 single-factor serum

[0063] 6 strains of H9N2 subtype AIV isolated in 2009 (including the vaccine backup strain A / Chicken / Shangong / SG2 / 2009 clearly involved in this paper), and 5 strains of H9 subtype avian influenza virus isolated before 2008, a total of 11 viruses , to prepare oil emulsion inactivated vaccines respectively. The 4-week-old SPF chickens were immunized with the above vaccines, raised in an isolated feeder to prepare single-factor serum, and immunized twice with an interval of 2 weeks. Blood was collected 14 days after the second immunization, and positive serum was prepared respectively.

[0064] 2. Crossover HI test

[0065] 11 strains of viruses were used as antigens, and 4 units of viruses were prepared respectively, and cross-HI tests were performed with 11 single-factor sera to detect the reactivity of the HA antigens of these H9N2 viruses. The experiment was repeated three times, and the averag...

Embodiment 3

[0081] 1. Vaccine Preparation and Immune Challenge

[0082] In order to determine the immunogenicity and vaccine protection effect of the vaccine strain virus, an oil emulsion inactivated vaccine was prepared, and 20 3-week-old SPF chickens were immunized. At the same time, 6 SPF chickens were immunized with PBS as a blank control. Serum was collected three weeks after immunization and tested. HI antibody levels to the virus. Immunization chicken is divided into two groups, every group 10, and I group carries out challenge virus (intravenous injection) with vaccine virus A / Chicken / Shangong / SG2 / 2009 (H9N2) (CK / SD / SG2 / 2009 for short) of the present invention , Group II was challenged with A / Chicken / Henan / L3 / 2008 (H9N2) (CK / HN / L3 / 2008 for short), and the dose was 100ul, (100ul contained 10 5 EID 50 ). Three and five days after the challenge, the throat cotton and cloacal cotton were collected from each chicken in the test group and the control group, and immediately placed in ...

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Abstract

The present invention relates to the field of animal virology, and provides a recombinant chicken-origin H9N2 avian influenza virus vaccine strain and a method for isolation, identification and purification of the strain. The invention further relates to a research of biological characteristics of the strain, especially to a research of characteristics of the strain adopted as the vaccine strain,and an evaluation of immune effects of the strain on SPF chickens. The preservation number of the strain is CCTCCNO:V201030. According to the present invention, the antigen variation conditions of the virus strain and other isolated virus strains are represented from the molecular level; after the virus strain is prepared into the vaccine, the prepared vaccine is adopted to immunize the 4 week old SPF chickens, with the protection effect analysis of the homologous H9 influenza wild virus strain and the heterologous H9 influenza wild virus strain, the results show that the influenza virus strain can be adopted as the spare vaccine strain of H9 subtype avian influenza. With the present invention, the spare vaccine strain is provided for prevention of the avian influenza outbreak by using the vaccine, the molecular biology technology program is provided for screen of the avian influenza virus vaccine strain, the molecular biology background is provided for study of the mechanism of animal infection by the avian influenza, and the important public health significance is provided.

Description

technical field [0001] The present invention relates to the field of animal virology. The present invention provides a H9N2 avian influenza virus vaccine strain, which is actually a naturally recombined H9N2 avian influenza virus strain of chicken origin, and provides a screening process of the vaccine strain and its Analysis of immune effect on SPF chickens after immune protection, especially after making fire-fighting vaccine. Background technique [0002] Avian influenza virus (AIV) belongs to the genus Influenzavirus 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 RNA virus 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 Pathogenic Avian Influenza virus, HPAlV...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/00A61K39/145A61P31/16G01N33/86C12R1/93
Inventor 许传田张秀美胡北侠鲁梅颜世敢杨少华张琳
Owner INST OF ANIMAL SCI & VETERINARY MEDICINE SHANDONG ACADEMY OF AGRI SCI
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