If cultures without full CPE are used, the supernatant shall be removed, cells shall be scraped from the surface of the well or flask and placed in a sterile 1,5 ml tube for RNA extraction and RT-PCR.

Treatment of all inocula with antiserum to IPN virus (a virus which in some parts of Europe occurs in 50 % of fish samples) aims at preventing CPE due to IPN virus from developing in inoculated cell cultures.

After exposure to test chemicals with and without an appropriate metabolic activation system, cell cultures are treated with spindle inhibitors such as colchicine to accumulate cells in a metaphase-like stage of mitosis (c-metaphase).

III.5.1. Subcultivation shall be carried out between days 13 to 15. Two hundred and twenty-five >ISO_7>ě>ISO_1>l of culture supernatant shall be added to wells containing fresh actively growing SHK-1 cells in 12-well plates and incubated at 14 ± 2 °C for up to 18 days.

Where practical difficulties arise (e.g. incubator breakdown, problems with cell cultures, etc.) which make it impossible to inoculate cells within 48 hours after the collection of the tissue samples, it is acceptable to freeze the supernatant at -80 °C and carry out virological examination within 14 days.

Where practical difficulties arise (e.g. incubator breakdown, problems with cell cultures, etc.) which make it impossible to inoculate cells within 48 hours after the collection of the tissue samples, it is acceptable to freeze the supernatant at -80 °C and carry out virological examination within 14 days.

Information shall be provided on the use of any raw materials (e.g., cytokines, growth factors, culture media) or of possible ancillary products and medical devices e.g., cell sorting devices, biocompatible polymers, matrix, fibres, beads in terms of bio-compatibility, functionality as well as the risk of infectious agents.

It is recommended that the biosecurity unit for ASF virus work should be supported by areas where the ASF virus is not manipulated. These other areas should be available for the preparation of glassware and media, the maintenance and preparation of non-infected cell cultures, the processing of sera and serological testing (other than methods using live ASF virus), and the provision of administrative and clerical support.

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.

The requirements laid down in table 1 must be fulfilled in any laboratory where the ASF virus is to be amplified by replication in cell cultures. However, post-mortem examinations, processing of tissues for DIFT or PCR and serology using inactivated antigens, may be carried out at a lower containment level, provided that the minimum requirements of table 1 are fulfilled, basic hygiene is used and post-operational disinfection with safe disposal of carcases, tissues and sera are carried out.

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 HAD technique is recommended for the identification of ASF virus isolates due to its high sensitivity and specificity. HAD is based on the capability of the ASF virus to replicate in pig macrophages and induce haemadsorption in the presence of pig erythrocytes. A characteristic `rosette` of erythrocytes develops around the infected macrophages. However, a small number of field ASF virus strains may not induce haemadsorption, but they produce a cytopathic effect. These strains may be specifically identified using the DIF test on the sediments of the cell cultures or by PCR.

the special cell culture in which the

collagen synthesis in bone cell culture;

1 ml 1: cell culture infective dose 50 %

uninfected SHK-1 cell culture or other susceptible cell culture (negative control),

ISAV-infected SHK-1 cell culture or other susceptible cell culture (positive control).

methods for maintenance of cell culture, if applicable,

Cidofovir Inhibition of Virus Multiplication in Cell Culture

A cell culture study should be performed in human cell or tissue cultures of different organs.

1 ml 1: cell culture infective dose 50 % 2: egg infective dose 50 %

* 50 % cell culture infectious dose 1 produced in chick embryo cells.

It is particularly important that cell culture conditions ensure a cell cycle time within the normal historical range of the cells or cell line used.

The ratio between inoculum size and volume of cell culture medium should be about 1:10.

Autolysed samples can be cytotoxic to the cell culture and consequently of limited use.

FeLV recombinant Canarypox virus (vCP97) ≥ 10 7.2 CCID50 (cell culture infective dose)

1 ml 1: cell culture infective dose 50 % For a full list of excipients, see section 6.1.

Each cell culture is harvested and processed separately for the preparation of chromosomes.

"cell culture" shall mean the in-vitro growth of cells derived from multicellular organisms.

No differences were observed between the cell-culture and the comparator egg-derived vaccine.

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

Additionally, live measles vaccine and live mumps vaccine are produced in chick embryo cell culture.

FeLV recombinant Canarypox virus (vCP97)......... ≥ 10 7.2 CCID50 (cell culture infective dose 50 %)

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).

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

number of cell cultures;

number of cell cultures,

Two further cell culture passages may be necessary in order to detect small amounts of the virus in the sample.

1 ml 1: cell culture infective dose 50 % 2: egg infective dose 50 % For a full list of excipients, see section 6.1.

Lamivudine has been shown to be highly synergistic with zidovudine, inhibiting the replication of HIV in cell culture.

Atazanavir has activity against HIV-1 Group M subtype viruses A, B, C, D, AE, AG, F, G, and J isolates in cell culture.

virus infectivity assay, (ii) serum toxicity controls, (iii) uninoculated cell culture controls, (iv) reference antisera.

Lamivudine in combination with zidovudine exhibits synergistic anti-HIV activity against clinical isolates in cell culture.

isolation and identification of ISAV in cell culture from a single sample from any fish on the farm as described in Part III;

Incorporation of additional ETVr amino acid changes rtT184, rtS202 or rtM250 decreases entecavir susceptibility in cell culture.

number of cells per culture,

isolation and identification of ISAV in cell culture from at least one sample from any fish on the farm as described in Part III,

Sera for the test are diluted 1/4 in serum-free cell culture medium with the addition of 100 IU/ml neomycin or other suitable antibiotics.

Epoetin alfa did not show any changes in bacterial and mammalian cell culture mutagenicity tests and an in vivo micronucleus test in mice.

Erythropoietin did not show any changes in bacterial and mammalian cell culture mutagenicity tests and an in vivo micronucleus test in mice.