Division III Procedure for titration to verify the susceptibility of the cell cultures to infection

When titration to verify the susceptibility of the cell cultures to infection as referred to in point I.5.3 is carried out, the procedures set out in the following paragraphs of this point shall be followed.

At least two VHSV isolates and one isolate of IHNV shall be used. The isolates shall represent the major group of viruses within F1Europe, namely for VHSV one pathogenic isolate from rainbow trout in freshwater and one marine isolate pathogenic for turbot, and for IHNV one rainbow trout pathogenic strain from Europe. Well-defined isolates F2... shall be used. Batches of virus in low cell culture passage numbers shall be propagated in cell culture flasks on BF-2 or RTG-2 cells for VHSV and on EPC or FHM cells for IHNV. Cell culture medium with at least 10 % serum shall be used. Low MOI for inoculation (< 1) shall be used.

At total CPE, the virus shall be harvested by centrifugation of cell culture supernatant at 2 000 × g for 15 minutes, filter sterilised through 0,45 μm membrane filter and distributed in labelled cryotubes. The virus shall be kept at – 80 °C.

1 week after freezing, three replicate vials with each virus shall be thawed under cold water and titrated on their respective cell lines. At least every 6 months, or if it is suspected that the susceptibility of a cell line has decreased, each virus isolate shall be thawed and titrated.

Titration procedures must be described in detail and the same procedure followed each time.

Titration by end point dilution shall include at least six replicates at each dilution step. The titres shall be compared with previously obtained titres. If the titre of any of the three virus isolates drops by a factor of 2 logs or more, compared with the initial titre, the cell line shall no longer be used for surveillance purposes.

If different cell lines are kept in the laboratory each line shall be examined separately.

Records shall be kept for a period of at least 10 years.