Cultures of established cell lines as well as primary cell cultures may be used.

Established cell lines or cultures of primary cells are used, e.g. Chinese hamster cells and human lymphocytes.

If no CPE has developed after the primary incubation for 7 to 10 days, subcultivation is performed to fresh cell cultures utilising a cell area similar to that of the primary culture.

Aliquots of medium (supernatant) from all cultures/wells constituting the primary culture are pooled according to cell line 7 to 10 days after inoculation.

Antibiotic-treated organ suspension is inoculated into cell cultures in two dilutions, i.e. the primary dilution and, in addition, a 1:10 dilution thereof, resulting in final dilutions of tissue material in cell culture medium of 1:100 and 1:1000, respectively, (in order to prevent homologous interference).

Virus isolation is based on the inoculation of sample material on susceptible primary cell cultures of porcine origin, monocytes and macrophages cells. The preferred samples for isolation of the ASF virus are whole blood and leucocytes obtained from non-coagulated blood samples or the organs referred to in section A(1). If the ASF virus is present in the sample, it will replicate in the cells and a characteristic cytopathic effect will be produced in the infected cells.

III.5.3. If bacterial contamination occurs in the primary culture, the test must be set up again using the tissue homogenate stored at - 80 °C. Prior to inoculation the tissue homogenate is centrifuged at 4000 x g for 30 minutes at 0 to 6 °C and the supernatant is filtered at 0,22 >ISO_7>ě>ISO_1>m. If bacterial contamination occurs during the subcultivation step the supernatant shall be filtered at 0,22 >ISO_7>ě>ISO_1>m, inoculated onto fresh cells and incubated for a further 14 to 18 days.

The in vitro chromosome aberration test may employ cultures of established cells lines, cell strains or primary cell cultures.

A variety of cell lines, strains or primary cell cultures, including human cells, may be used

Samples are inoculated into cultures of primary bovine thyroid cell cultures using at least three tubes per sample.

Alternatively aliquots of 10 % of the medium constituting the primary culture is inoculated directly into a well with fresh cell culture (well to well subcultivation).

for viruses and viroids, cell culture studies using purified infective virus and primary cell cultures of mammalian, avian and fish cells

Data as outlined under point 5.1.1 and cell culture studies using purified infective virus and primary cell cultures of mammalian, avian and fish cells

Virus isolation is based on the inoculation of sample material on susceptible primary cell cultures of porcine origin, monocytes and macrophages cells.

In addition, raltegravir inhibited viral replication in cultures of mitogen-activated human peripheral blood mononuclear cells infected with diverse, primary clinical isolates of HIV-1, including isolates resistant to reverse transcriptase inhibitors and protease inhibitors.

The simultaneous inoculation of a porcine cell line and one of the above-mentioned tissue culture systems (preferably primary bovine thyroid cells) is a useful guide as to whether vesicular samples contain swine vesicular disease virus or foot and mouth disease virus, as the former will only grow in cells of porcine origin.

Indinavir at concentrations of 25 to 100 nM mediated 95 % inhibition of viral spread in cultures of mitogen– activated human peripheral blood mononuclear cells infected with diverse, primary clinical isolates of HIV− 1, including isolates resistant to zidovudine and non– nucleoside reverse transcriptase inhibitors (NNRTIs).

Microbiology Indinavir at concentrations of 50 to 100 nM mediated 95 % inhibition (IC95) of viral spread (relative to an untreated virus– infected control) in human T– lymphoid cell cultures and primary human monocytes/ macrophages infected with HIV− 1 variants LAI, MN, RF, and a macrophage– tropic variant SF– 162, respectively.