Growth and infectivity assays of the Israeli vaccine strain of fowl poxvirus in chicken embryo fibroblasts.
The Israeli vaccine strain of fowl poxvirus grows efficiently in chicken embryo fibroblasts but not in cell lines derived from monkey kidney or human fibroblasts. We developed two assays for the titration of the infectivity of this virus in secondary cultures of chicken embryo fibroblasts. The first is a focus assay, in which minimum essential medium and SeaKem ME agarose were used for the overlay media. Under these conditions, clear virus foci appeared after 5 days of incubation at 37 C. The second assay is a semiautomatic colorimetric test based on the ability of live cells in culture to reduce the yellow tetrazolium salt 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT; thiazolyl blue) to its formazan derivative. The reagent was added to infected chicken embryo fibroblasts in 96-well plates 10 days after infection. The formazan formed during 2 hr was extracted with dimethyl sulfoxide, and its absorbance was read by an automatic microplate spectrophotometer. A good correlation of the infectivity titers of the virus was obtained by the two methods.
Hashavya
Saar
S
Department of Virology, Hebrew University-Hadassah Medical School, Jerusalem, Israel.
Barchichat
Sabrina
S
Katz
Ehud
E
D000818:Animals; D002478:Cells, Cultured; D002642:Chick Embryo / Q000166:cytology; D003124:Colorimetry / Q000379:methods / Q000662:veterinary*; D003470:Culture Media; D003584:Cytological Techniques / Q000662:veterinary; D005347:Fibroblasts / Q000821:virology*; D005562:Formazans; D005587:Fowlpox virus / Q000254:growth & development* / Q000276:immunology / Q000472:pathogenicity*; D013778:Tetrazolium Salts / Q000378:metabolism; D013844:Thiazoles / Q000378:metabolism; D013997:Time Factors; D010948:Viral Plaque Assay / Q000662:veterinary; D014765:Viral Vaccines
D003470:Culture Media; D005562:Formazans; D013778:Tetrazolium Salts; D013844:Thiazoles; D014765:Viral Vaccines; C022616:thiazolyl blue