Cholesterol affects African swine fever virus infection.

African swine fever virus (ASFV) enters cells by receptor mediated endocytosis and requires a fusion event between the viral envelope and the limiting membrane of the endosome at low pH. In order to investigate the role of cholesterol in the early stages of ASFV infection, we have studied the effect of the removal of cell and viral membrane cholesterol by cholesterol oxidase treatment of cells and virions, as well as the effect of some inhibitors of cholesterol synthesis on the infectious pathway. In addition, we have investigated viral infection in cholesterol-depleted Vero cells. Both cholesterol-depleted and cholesterol oxidase-treated Vero cells were unaltered in their ability to bind or internalize the virus, but were blocked in ASFV fusion and subsequent virus replication. Our results indicate that ASFV infection is affected by cholesterol in the target membrane.

An antimicrobial abietane from the root of Plectranthus hereroensis.

A new abietane diterpene, 16-acetoxy-7 alpha, 12-dihydroxy-8,12-abietadiene-11,14-dione, has been isolated from the acetone extract of the root of Plectranthus hereroensis and its structure established by spectroscopic means. This compound showed antibacterial activity against Staphylococcus aureus and Vibrio cholerae, and antiviral activity against Herpes simplex type II.

Entry of African swine fever virus into Vero cells and uncoating.

African swine fever virus (ASFV) enters Vero cells by adsorptive endocytosis [Valdeira, M.L., Geraldes, A., 1985. Morphological study on the entry of African swine fever virus into cells, Biol Cell. 55, 35-40]. Electron microscopy of a lysosomotropic drug-controlled penetration indicated that this step takes place in the endosomes, after fusion between the viral envelope and the limiting membrane of the endosome. Inhibition studies with colcemid, cytochalasin B, sodium azide, dinitrophenol, lysosomotropic weak bases, and the ionophore monensin, showed that the virus uptake is largely independent of cytoskeletal and lysosomal function, but dependent on oxidative phosphorylation. Some protease inhibitors inhibited viral replication at an early step, indicating that the initiation of infection depends on a viral proteolytic cleavage.

Effect of chloroquine on African swine fever virus infection.

When 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 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.

Morphological study on the entry of African swine fever virus into cells.

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.

Evidence for an acid phosphatase in African swine fever virus.

An acid phosphatase activity has been detected in purified preparations of African swine fever virus. Purified viral cores obtained after Nonidet-P40 and 2-mercaptoethanol treatment of the virus retained the activity as assayed with nitrophenyl phosphate as substrate at pH 5. Enzyme cytochemistry by electron microscopy showed that the acid phosphatase activity is localized mainly inside the core of the virion. The molecular weight and the isoelectric point of the virus acid phosphatase activity confirmed that it was distinct from the host cellular enzyme.

Fusion activity of African swine fever virus towards target membranes: lipid dependence and effect of dehydrating agents.

Fusion of African swine fever virus (ASFV) with model membranes was monitored by a fluorescence dequenching assay. ASFV was able to fuse with liposomes of various compositions. Fusion was more extensive with liposomes made of negatively charged phospholipids, and reduced in the presence of the neutral phospholipid phosphatidylcholine (PC). Fusion activity of ASFV was pH-dependent, the extent of fusion increasing with decreasing pH for all target membranes. These results are consistent with the in vivo characteristics of fusion of ASFV with the endosome membrane. An increase in fusion activity was obtained with dehydrating agents, similarly to what occurs with other lipid-enveloped viruses. Dehydrating agents such as dimethylsulfoxide and dimethylsulfone greatly enhanced fusion, the effect of dimethylsulfone being more pronounced than that of dimethylsulfoxide. Poly(ethylene glycol) also potentiated ASFV fusion activity, and the effect of this polymer was found to be dependent on its molecular weight. These results stress the importance of dehydration and hydrophobic interactions on the early events of viral penetration into target cells.

Catechols from abietic acid synthesis and evaluation as bioactive compounds.

Catechols from abietic acid were prepared by a short and good yielding chemical process and further evaluated for several biological activities namely, antifungal, antitumoral, antimutagenic, antiviral, antiproliferative and inhibition of nitric oxide. Their properties were compared with those of carnosic acid (6), a naturally occurring catechol with an abietane skeleton and known to possess potent antioxidant activity, as well as anticancer and antiviral properties. From all the synthetic catechols tested compound 2 showed the best activities, stronger than carnosic acid.