Study Of FMDV Large-scale Culture And Virus Protectant

Foot-and-mouth disease virus (FMDV) infection results in Foot- and-mouth disease (FMD), a widespread and highly contagious anthropo zoonosis, which greatly threaten cloven-foot animals and human health. As a kind of Picornaviridae ,FMDV can infect many animals, such as cattle, sheep, goats, buffalos, deer, antelope, and pigs. Spreading swiftly served as a character to the FMD endemic.The culture of BHK-21 cell and FMDV were studied on roller bott- le.BHK-21 cell could atach to roller bottle and grow well.Changed roller speed 2r/min, reducing culture volume and treating roller bottle with calf serum could improve the ataching efect of BHK-21 cell. At the PH7.2 of cells culture fluid,the BHK-21 cell density reaching hightest.The BHK-21 cell density reaching 12×105 cell/ml inoculation dose reaching 0.33-0.67%; cell density reaching 6×105 cell/ml inoculation dose reaching 0.64-1.34%; cell density reaching 3×105 cell/ml inoculation dose reaching 0.167% in this system. After infected with FMDV, the cell on roller bottle appeared pathological changes. The best inoculation way is single inocula- tion in 24 hours at 37℃.Select gelatin,cane-suger,trehalose,urea,L-arginine,antiscorbic-acid, sorbitol et.al make up match.prepare to 15 groups virus protectant The test applied 15 groups virus protectant toBHK-21 cell lines containing virus. Through cells morpHology observation, screened a virus protectant and determined its match 1:8. Employed directim munofluorescence in order to screen models about best cells density,best inoculation dose,best inoculation and harvest way.Th rough establishing virus proliferation curve about control and experime- nt(containing virus preserve),we found that it was not obviously d ifferent to detect virus proliferation by direct immunofluorescence,however the fluorescence plaque of experiment was lighter and denser than that of control,when in the same titres.Further,we employed living cells dyeing excluding,found that at 60 hour,living cells number containing virus of ex periment were 1.22% that of control,however living cells ratio was with one accord.we also confirmed the above result by employing MTT displays.In vaccine production,virus protein concentration of experiment was 22% higher than that of control.Apllying virus preserver into vaccine production of FMDV bovine testicle cell vaccine,we found as the result of freezing and thawing again and again showed ,6 times by repeating freezing and thawing in the control was critical point. If it was over6, the control would not qualify whereas the experiment would qualify exceeding 10 times. Cryopreservation for a long time showed that control would qualify in 3 months whereas experiment would qualify in 5 months.the result of bearing heat showed control would qualify in 24 hours at 37℃, whereas experiment would qualify in 48-60 hours at 37℃.