Viral Assay
A virus and virus replication technology, applied to animals with reduced sensitivity, to determine the sensitivity of animals to viruses, and to detect virus replication in tissue samples.
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Embodiment 1
[0249] Example 1 Preparation of tissue samples
[0250] Chicken skin (from the breast area under the wings), thumbs (winglets) and feather sacs were taken from 12-day-old chicks and placed in supplements supplemented with penicillin (100U / ml) and streptomycin at room temperature. (100 μg / ml) in PBSA. Samples were processed within 1.5 hours of removal from the chickens and infected with the RP8 strain of influenza A virus. The skin sample was cut into small pieces of about 2mm, and the feather capsule was cut into small pieces of 1cm, and the medulla inside was squeezed out. The medulla in the vesicle was about the same size as the skin tissue block. Individual tissue pieces were plated in 96-well plates containing 200 μl PBSA.
Embodiment 2
[0251] Embodiment 2 virus test
[0252]Use viral growth medium (VGM) containing 0.3% bovine serum albumin (BSA), 10 mM Hepes, 2 mM glutamate, supplemented with penicillin (100 U / ml), streptomycin (100 μg / ml), amphotericin B (0.005 μg / ml) and 5 μg / ml insulin in Earls' modified Eagle medium to serially dilute the virus of the PR8 strain of influenza A virus (stored in the allantoic fluid of eggs conceived from 10-day-old chickens) 10-fold to 10 -5 (1000TCID50 infectious dose). Remove PBSA, add 200 μl virus to each well, at 37 °C, containing 5% CO 2 Incubate in a humid atmosphere for 1 hour, then remove the virus, add 200 μl of VGM containing 5 μg / ml insulin, at 37 ° C, containing 5% CO 2 Continue to incubate for 1 hour or 48 hours under a humidified atmosphere.
[0253] The virus supernatant was removed, the tissue was washed with 200 μl of PBSA, and after being spun through the QIAshredder column, RNA was isolated according to the instructions of the Qiagen RNeasy kit. RNA ...
Embodiment 3
[0254] Example 3 Determination of virus susceptibility in the chicken embryo model
[0255] 4-day-old specific pathogen-free (SPF) embryos were injected with RCAS virus, which expressed EGFP&shRNA, in which the shRNA targeted the PB1 gene of influenza A virus (PB1-2257-5'gatctgttccaccattgaa3' (sequence number: 3). In egg Make a small hole with the blunt end of the sac, exposing the air sac membrane, embryo and vasculature. Use a microcapillary to remove a few microliters of blood from the vessels, followed by 1-2 μl of RCAS virus (titer about 10 8 / ml) was injected from another blood vessel. Double seal the egg with two small strips of parafilm, place the parafilm over the opening, and seal the parafilm to the surface of the egg using a slightly heated scalpel blade. Eggs continued to be cultured until day 9.
[0256] Embryos were removed from eggs and EGFP was detected by fluorescence using a dissecting microscope. Embryos containing the highest density of EGFP expression ...
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