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The early interactions between African swine fever virus (ASFV) and monkey kidney cells in culture, and the effect of chloroquine were studied by electron microscopy. Our results indicate that ASFV uptake occurs by endocytosis: after attachment to the cell surface, the virions were seen in coated pits and were internalized by endocytosis in endosomes and finally in lysosomes. Virions in coated vesicles were never seen. All these steps were completed in about 15 min. Direct penetration of viruses through the plasma membrane was never observed. In order to elucidate the participation of an acidic intracellular compartment in the penetration of ASFV, we studied the effect of chloroquine, a lysosomotropic drug known to increase the pH of acidic intracellular vacuoles and to inhibit ASFV infection. In the presence of this drug there were no apparent alterations on binding, endocytosis and intracellular distribution of the viral particles. The main effect of chloroquine was to retain the virions in lysosomes. When the drug was removed from the medium, the viral particles disappeared and images of binding of viral membranes with the membranes of the intracellular vacuoles were obtained, suggesting that the inhibited step is the uncoating of the virus. Viral fusion with the plasma membrane was obtained when the medium was acidified to pH 5-6. These results suggest that ASFV enters the cells by adsorptive endocytosis and that the uncoating process takes place intracellularly in a way similar to that described for Semliki Forest virus and other enveloped viruses.

SUMMARYWhen present during the whole infective cycle, the lysosomotropic drug, chloroquine, inhibited cytopathic changes and production of African swine fever virus (ASFV) in Vero cells. This inhibition decreased when the drug was added from 1 h to 4 h after infection. Chloroquine had no direct effect on the virus nor on viral adsorption and internalization. Electron microscopy showed that, in the presence of the drug, the virions were retained in large vacuoles having a lysosomal appearance. This inhibition was fully reversible, even when the drug was removed as late as 72 h after infection. The results support the hypothesis that ASFV enters the cells by adsorptive endocytosis and not by fusion with the plasma membrane.

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