Focus formation by rubella virus in rabbit kidney (RK-13) cell cultures and its application to the virus titration

Abstract

SummaryFour different strains of rubella viruses formed foci under agar overlay medium on 1~K-13 cells. This focus formation method may be more useful in the quantitative titration of rubella virus.The quantitation of rubella virus infectivity is based on the interference with other viruses, cytopathic effect on several cell systems, plaque formation or detection ef hemadsorption-negative plaques. However, these methods are relatively complicated and require long incubation periods. The present author observed a focus formation of rubella virus during efforts to reproduce the plaque titration of the M33 strain of rubella virus on R K -t 3 cells using plastic plates. The M33 strain of r~$ella virus was kindly supplied by Dr. 5{. Ohtawara, and the To-336 wild (2) and Cendehill strains of rubella viruses were supplied by Dr. R. Kono, N I H , Murayama, Tokyo, Japan. An attenuated strain of To-336 (3) was purchased from Takeda Pharmacy Co. LTD., Hikari, Yamaguchi, ,Japan. In the present experiments, viruses were groom in 1~K-13 and Vero cells. The seed viruses were prepared from infected tissue culture fluids on the 5th day of infection. RK-13 cells maintained on Eagle's ME~I supplemented with 5 per cent beat inactivated fetal bovine serum (FBS) and Kanamycin (60 mg/ml) were seeded at the dose of 1.5 × 10510.5 ml/well into the wells of semi-micro plastic tray (Limbro, 16 mm in diameter, 24 wells), and incubated at 37 ° C in a 5 per cent C02 incubator. Before the infection, cells were washed with Hanks' BSS, and inoculated with 0.1 ml/ well of serial ten-fold dilution of the virus in 5 per cent FBS-Tris LH (0.5 per cent Lactalbumin hydrolysate, 0.05 M Tris-HC1 in Hanks' BSS p H 7.4). After adsorption for 1 hour at 37 ° C, the inoculum was removed and the cell layer was overlaid with 0.5 ml of agar overlay medium (10 per cent FBS, 0.22 per cent NaHCOa, 0.8 per cent noble agar in Eagle's MEM).