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Separation identification and purification process for chicken source H9N2 avian influenza virus strain and uses thereof

A technology of bird flu virus, strain, applied in the field of animal virology

Inactive Publication Date: 2010-09-29
INST OF ANIMAL SCI & VETERINARY MEDICINE SHANDONG ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

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, decreased egg production of laying hens, and makes chickens prone to secondary severe respiratory diseases, affecting poultry production performance

Method used

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  • Separation identification and purification process for chicken source H9N2 avian influenza virus strain and uses thereof
  • Separation identification and purification process for chicken source H9N2 avian influenza virus strain and uses thereof
  • Separation identification and purification process for chicken source H9N2 avian influenza virus strain and uses thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Virus isolation and identification

[0029] A recombinant chicken source H9N2 avian influenza virus strain A / Chicken / Henan / L2 / 2008 (H9N2), the isolation and identification methods of the virus are as follows:

[0030] 1. Treatment of disease materials and inoculation of chicken embryo allantoic cavity

[0031] Take the internal organs (liver, kidney, spleen, etc.) of the diseased chicken and add sterilized saline to grind into a homogenate, freeze and thaw 3 times, centrifuge at 8000 revolutions per minute (rpm) for 10 minutes, discard the upper layer of fat, and absorb the middle liquid . Add penicillin and streptomycin and treat at 4°C for 1 hour, centrifuge again at 8000 rpm for 10 minutes, take the supernatant and inoculate 11-day-old SPF chicken embryos through the allantoic cavity, 0.2ml / piece, and culture them in an incubator for 72 hours. Observe twice a day, discard the contaminated embryos within 24 hours, and collect the allantoic fluid of the remaining chi...

Embodiment 2

[0041] Viral HA antigenicity analysis

[0042] experimental method:

[0043] 1. Preparation of H9 single-factor serum

[0044] Eleven isolated strains of H9N2 subtype AIV and A / Chicken / Henan / L2 / 2008 involved in the present invention were used to prepare oil emulsion inactivated vaccines respectively. The one-month-old SPF chickens were immunized with the above vaccines, and raised in an isolated feeder to prepare single-factor serum. A total of two immunizations were performed with an interval of 2 weeks. Blood was collected 14 days after the second immunization, and positive serum was prepared respectively.

[0045] 2. Crossover HI test

[0046] Using the above-mentioned 12 strains of viruses as antigens, 4 units of viruses were prepared respectively, and the cross-HI test was carried out with the above-mentioned 12 kinds of single-factor sera to detect the reactivity of the HA antigens of these H9N2 viruses. Experiments were repeated three times. The average value of th...

Embodiment 3

[0059] Determination of the whole genome sequence of the virus

[0060] experimental method:

[0061] 1. Synthesis of RT-PCR primers

[0062] Referring to the published H9 subtype AIV sequence data, RT-PCR primers for 8 gene segments (PB2, PB1, PA, HA, NA, NP, M, NS) of the AIV genome were designed and synthesized (see Table 2).

[0063] Table 2 Primer sequences for amplification of each gene fragment of avian influenza virus

[0064]

[0065]

[0066] 2. Extraction of viral RNA

[0067] Toxic chick embryo allantoic fluid was used as the material for RNA extraction, and the operation was performed according to the instructions of the Trizol kit (Invitragen).

[0068] 3. RT-PCR of each gene

[0069] Using TAKARA's PrimeScript TM One Step RT-PCR Kit, the reaction system is 50ul: RNase FreedH 2 O 30.5 μl, 10×Buffer 5 μl, Enhancer Solution 1 μl, dNTP Mixture 2 μl, RNase Inhibitor 1 μl, PrimeScript TM RTase 0.5 μl, EX Taq TM HS 1 μl, RNA sample 6 μl, upstream and down...

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Abstract

The invention relates to the field of animal virology, and provides separation identification and purification methods, biological characteristics and applications in biology of a recombinant fowl H9N2 avian influenza virus strain with preservation number being CCTCC NO:V200811; the differences between the virus and other separation strains are explained in term of molecular level; and the virus strain is proved to be capable of being used as candidate strain of avian influenza vaccines and as antigen of H9 subtype AIV hemoagglutination and hemoagglutination inhibition test (HA-HI). The strain has significance in knowing the molecular evolution and antigenic variation of fowl H9N2 avian influenza virus in China.

Description

technical field [0001] The invention relates to the field of animal virology. The invention provides a method for isolating, identifying and purifying a recombinant chicken-origin H9N2 avian influenza virus strain, its biological characteristics, and its application in biology. 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) and low pathogenic AIV (LowPathogenic Avian Influenza virus, LPAIV) according to its pathogenicity. type-based. [0003] Since the H9N2 subtype of AIV...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N7/00C12N7/02G01N33/569C12Q1/70C12Q1/68A61K39/145A61P31/16
Inventor 黄艳艳胡北侠张秀美曹三杰文心田徐栋路希山
Owner INST OF ANIMAL SCI & VETERINARY MEDICINE SHANDONG ACADEMY OF AGRI SCI
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