Concentration du virus de l'hepatite a sur des membranes de cellulose

Abstract

Viral hepatitis type A is the most prevalent waterborne disease in U.S.A. and the number of cases has been increasing in recent years (Melnick et al., 1978). Till now, few works were performed on Hepatitis A Virus (HAV) concentration in water (Hejkal et al., 1982, Elkana et al., 1983). In this paper the filter adsorption-elution method, widely used with other viruses (Wallis and Melnick, 1967; Fields et Metcalf, 1975; Smith and Gerba, 1982), was selected to concentrate HAV from experimentally-contaminated distilled water. Filtration was performed through two cellulose membranes (RAWP 04700 and HAWP 04700 Millipore). Eluate or filtrate viruses were precipitated by polyethylene glycol 6000. HAV is detected by solid phase radioimmunoassay according to a previously described method (Deloince et al., 1982). Figure 1 shows the results of adsorbing HAV at different pH levels. Analysis of variance pointed out that, for the pH levels between 3 and 4, the differences were not significant ( P = 0.05). Table 1 shows the effects of pH and number of passages of 3% beef extract or 0.2 M glycin buffer on HAV elution. For both eluents the differences in function of pH levels and number of passages of eluent through the membrane were not significant (variance analysis P = 0.05). On the other hand, the comparison between the two eluents pointed out the higher efficiency of beef extract ( t-test, P = 0.001). After this procedure, HAV replication occurred in human hepatocarcinoma cells PLC/PRF/5 (Fig. 2) inoculated according to a previously described method (Crance et al., 1983). As poliovirus (Sobsey et al., 1973), HAV adsorption is the most efficient at acid pH levels. Between pH 4.5 and 5, the remarkable adsorption decrease might be related to the inversion of the HAV electrostatic charges sign, i.e. to the occurrence of an isoelectric point. The HAV elution is different from the poliovirus one, the efficiency of which increased with pH (Wallis et al., 1972) and eluent passage numbers (Farrah et al., 1976). That might suggest that from pH 7.5, both the HAV particles and membranes have negative charges which give enough repulsive forces to allow a good elution efficiency. Improvement of HAV elution efficiency by beef extract is similar to a result obtained with poliovirus by Landry et al. (1978). This effect might be explained by the proteinous material presence in beef extract. HAV replication in cell cultures shows the biological properties preservation after this procedure.