A kind of serotype 4 group I poultry adenovirus strain and its application
An avian adenovirus and virus strain technology, which is applied in the directions of viruses, antiviral agents, viruses/phages, etc., can solve the problems of non-prevention group I avian adenoviruses and commercial vaccines without prevention group I avian adenoviruses, etc. rate effect
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Embodiment 1
[0015] Example 1 Isolation, cultivation and identification of group I poultry adenovirus CPHB03 strain of serotype 4
[0016] In 2015, laying hens in a chicken farm in Hubei became ill and some chickens died acutely. The autopsy revealed hydropericardium, brittle liver, enlarged kidney, and reticular hemorrhage in the gland and stomach.
[0017] Aseptically take the liver of sick and dead chickens, grind the liver with a mortar, add sterile saline to it, mix the sample, transfer it to a 15mL centrifuge tube, centrifuge at 3000rpm for 10min, and filter the supernatant through a 0.22μm filter to obtain the virus liquid, spare. The above-mentioned virus liquid was original times, 10 -1 、10 -2 、10 -3 、10 -4 、10 -5 、10 -6 、10 -7 、10 -8 Inoculate well-growing primary chicken embryo liver cells at different dilutions, culture at 37°C for about 120 hours, freeze the culture with the highest dilution of about 70% cytopathic changes, aliquot it, mark it as F1 generation virus l...
Embodiment 2
[0026] The virulence determination of the group I poultry adenovirus CPHB03 strain of embodiment 2 serotype 4
[0027] 1. Toxicity of CPHB03 strain to chicken embryo liver cells (virus content of CPHB03 strain)
[0028] Prepare chicken embryonic liver cells and spread 96-well cell culture plates at 37°C, 5% CO 2 After culturing for about 24 hours under certain conditions, the cell growth solution in the 96-well cell culture plate was discarded. The F5 generation CPHB03 strain virus solution was diluted 10 times with the cell maintenance solution, and 10 -6 、10 -7 、10 -8 、10 -9 Inoculate 96-well cell culture plate with diluted virus solution, 0.1mL / well, 6 replicate wells for each dilution, place at 37°C, 5% CO 2 Incubator cultivation. Seven days after inoculation, the 96-well cell culture plate was observed under a microscope, and the number of wells with and without cytopathy at each dilution was counted, and the virus content of the tested samples was calculated by the...
Embodiment 3
[0035] Example 3 Immunogenicity Evaluation of Group I Avian Adenovirus CPHB03 Strain of Serotype 4
[0036] To the virus content is 10 7.5 TCID 50 Formaldehyde solution with a final concentration of 0.1% (v / v) was added to 0.1 mL of CPHB03 strain virus liquid, and inactivated at 37° C. for 24 hours. Take 0.5mL of inactivated antigen to inoculate chicken embryo liver cells cultured for 16-24 hours, as the F1 generation; if the F1 generation has no cytopathic changes, inoculate chicken embryo liver cells cultured for 24 hours, as the F2 generation; No cytopathic changes were observed in the F2 generation after 168 hours of culture, indicating that the tested antigen was completely inactivated. Add Tween 80 to the inactivated antigen so that Tween 80 accounts for 4% of the total volume, and mix well to form the water phase; add Siben 80 and aluminum stearate to the white oil to make Siben 80 account for 6% of the total volume 1. The mass volume ratio of aluminum stearate to wh...
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