Cold-adapted attenuated strain of h9n2 subtype avian influenza and its application
An avian influenza and cold-adaptation technology, applied in inactivation/attenuation, antiviral agents, medical preparations containing active ingredients, etc. Effective control of influenza virus outbreaks and other issues
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Embodiment 1
[0026] Embodiment 1, preparation and preservation of virus strain
[0027] 1. Preparation of virus strains
[0028] Incubate SPF chicken embryos at 37°C for 10 days; then, dilute the SD / 01 / 10-WT strain with sterile PBS buffer to 4 hemagglutination units (HAU), and inoculate 3 to 5 chickens at a dose of 0.2ml / piece Embryos were placed in a 33°C incubator to incubate for 48 hours, and dead embryos within 24 hours were discarded; then, after 12 hours in a refrigerator at 4°C, the allantoic fluid of chicken embryos was collected, and 0.5% chicken red blood cells were used for hemagglutination test to preserve the hemagglutination The one with the highest titer of ETV was marked as the first generation.
[0029] 33°C to 25°C gradually lowering the temperature for continuous passage. For each passage, 3 to 5 10-day-old chicken embryo allantoic cavities were inoculated with 4 HAU viruses diluted in sterile PBS buffer, and incubated for 48 hours in a 4°C refrigerator. The allantoic ...
Embodiment 2
[0033] Embodiment 2, the identification of virus strain
[0034] The virus to be tested is H9AIV-SD / 01 / 10-CA strain or SD / 01 / 10-WT strain.
[0035] 1. Determination of virus PFU
[0036] 1. Cultivate a single layer of MDCK cells in a 6-well cell culture plate, remove the culture medium in the well, wash the cells with PBS buffer for 3 times, and discard the washing solution.
[0037] 2. Prepare ten 1.5mL EP tubes, add 900μL DMEM medium containing 100U / ml penicillin and 100μg / ml streptomycin to each tube; then, add 100μL virus stock solution of the virus to be tested into the first tube, mix thoroughly After uniformity, suck out 100 μL to the second tube, and operate to the tenth tube in turn, which is the virus dilution of each dilution, mark it, and store it on ice.
[0038] 3. Take the 6-well cell culture plate that completed step 1, inoculate the virus dilution (200 μL per well) obtained in step 2, and inoculate at 37°C, 5% CO 2 Incubate for 1 h in the incubator.
[003...
Embodiment 3
[0064] Embodiment 3, strain as the immunogenicity and protective evaluation of live vaccine
[0065] H9N2 subtype avian influenza virus challenge strains are: A / chicken / Shandong / sd01 / 2010, A / chicken / Beijing / 3 / 1999 (representative strain of genotype G02), A / chicken / Hebei / 0617 / 2007 (representative strain of genotype G51), A / chicken / Shandong / ZB / 2007 (representative strain of genotype G60), A / chicken / Hebei / YT / 2010 (representative strain of genotype G57) and A / chicken / Guangdong / 01 / 2011 (representative strain of genotype G68).
[0066] The experimental subjects were 1-week-old SPF Laihang chickens (purchased from Beijing Meria Weitong Experimental Technology Co., Ltd.).
[0067] Each challenge strain is carried out as follows experiment respectively:
[0068] Immunization group: Take 3 experimental subjects and immunize H9AIV-SD / 01 / 10-CA strain once (intranasal immunization, immunization dose is 10 6 EID 50 , immunization volume is 200μl), these 3 experimental subjects belong t...
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