Routine titration of foot and mouth disease virus suspensions by analytical ultracentrifugation. 1: Sedimentation method

Abstract

Infectivity and Complement Fixation (CF) tests are commonly used for the routine titration of Foot and Mouth Disease (FMD) virus suspensions. Only recently were techniques published for the routine determination of the virus concentration by the physical properties of the virions (Fayet et al., 1971; Barteling et al., 1974). These techniques are based on the separation of the virions from the culture fluid by sedimentation through a sucrose gradient, in a preparative ultracentrifuge. The ultraviolet absorption pattern of the tube content is recorded by a flow colorimeter. The virus concentration is estimated using either standard curves or direct caculation by the specific extinction coefficient (Bachrach et al., 1964). In our own attempts to develop a preparative ultracentrifugation technique for the routine titration of FMD virus suspensions, we had to deal with some problems such as remixing of the virus band at the end of the run. We therefore turned over to analytical ultracentrifugation methods. The manipulations are less complicated and the virus band is traced and measured while the rotor is spinning. Four samples are analyzed simultaneously and the scans are repeated to follow the move of the virus band. The sedimentation rate of the virus band, calculated from the repeated scans, helps to detect artifacts. The present paper describes the technique we developed for the routine titration of FMD virus suspensions, by the band sedimentation method, using an ultraviolet scanning analytical ultracentrifuge.