Patent DE-102008028406-A1 (DE)
Title: In vitro method for the propagation and increase of norovirus comprises growing uninfected Vero cells in tissue culture bottle, separately suspending norovirus positive patients stool, filtering suspension and sucking growing medium
Inventor: GROTGANS MARIA
IPC: C12N7/02
Filed: 2008-06-17
Abstract: In vitro method for the propagation and increase of norovirus, comprises (1) growing uninfected Vero cells in a tissue culture bottles; (2) separately suspending norovirus positive patients stool of genotype 1 and genotype 2; (3) filtering the suspension of the norovirus positive patients stool material; (4) sucking the growing medium from the cell culture bottle; (5) adding patient material filtrates into the respective cell culture bottle; (6) manually distributing the mixture; and (7) incubating the mixture in the incubator at 37[deg] and 5% carbon dioxide. In vitro method for the propagation and increase of norovirus, comprises (1) growing uninfected Vero cells (kidney cell line) from the African green monkey in a tissue culture bottles for a confluent cell layer (monolayer); (2) separately suspending norovirus positive patients stool of genotype 1 and genotype 2, further adding a suspending solution (for isodilution) containing amphotericin (50 mg bottles), penicillin (1662 IU/mg) and streptomycin (770 mu g/mg), where the above components are dissolved in a suitable medium; (3) filtering the suspension of the norovirus positive patients stool material, where the filtration comprises (a) a 20 ml disposable syringe, (b) separating the piston from the syringe body, (c) minifilter of 0.45 mu permeabilities, (d) cone of the filter in the bottom cone of the syringe body, (e) filter with the syringe body of an open tissue culture small tube, (f) filling the suspension of the patient material into the syringe body, (g) setting the pistons in the syringe body and carefully filtering the suspension and (h) for the respective patient materials/suspension, genotype 1 and genotype 2 a new disposable syringe filter and tissue culture small tube is used; (4) sucking the growing medium from the cell culture bottle by a pipette or a sucking devices; (5) adding patient material filtrates (500 mu l) into the respective cell culture bottle with uninfected Vero cells inoculated with a pipette, and further adding following substances (a1) non-essential amino acid (250 mu l), (b1) trypsin ethylenediaminetetraacetic acid (50 mu l), and (c1) fetal bovine serum (300 mu l); (6) manually distributing the mixture through easy here and there move of the respective cell culture bottle; (7) incubating the mixture in the incubator at 37[deg] and 5% carbon dioxide;(8) manually moving the cell culture bottle for over 6 hours with manual movement for quarter hour; (9) further incubating the cell culture bottle for 37[deg] and 5% carbon dioxide for 15 hour; (10) supplying enriched culture medium (8 ml) into the respective cell culture bottle with the patient material filtrate, after the incubation time;(11) culture medium enrichments contain minimum essential medium of bottles (500 ml), penicillin/streptomycin in a ratio of 1:100 with 10000 units, fetal bovine serum (10) and non-essential amino acid (1), where the result (12) of the above process is, after the point (9), after incubating 15 hours in the light microscope, provides a virus-induced cytophatic effect by giant cell formation (syncytia), and Real-time PCR results confirm the infection of these norovirus cells (arrangement as a graphical representation); (13) further cultivating the cell culture supernatant as the point 1 and 4; and (14) extracting cell culture supernatant from the infected tissue culture bottle with Vero cell using a pipette (500 mu l) and adding into a new uninfected tissue culture bottle, as like the point 5-12, where depending on the needs of a high titer of norovirus infection from cell culture supernatants of Vero cells, many so-called passages are possible.