Comparative ultrastructure of the new phleboviruses Arrabida and Alcube from Portugal and Toscana phlebovirus, ISS Phl.3 strain

Introduction: Phlebovirus genus (family Phenuiviridae), consists of 10 species of viruses composed by different serologically related viruses. Toscana virus (which belongs to Sandfly Fever Naples Virus- SFNV- species complex) is neurotropic and its presence was reported for the first time in Portugal in 1983. An entomological survey conducted in 2007-2008, in Arrabida region, in south Portugal detected two new phleboviruses of unknown pathogenic potential, provisionally named Arrabida and Alcube ascribed to SFNV and Salehabad Virus (SALV) species, respectively. Apart from a previous characterization of the Arrabida virus [ 1 ] these viruses, including the Toscana virus, have not been previously studied at the ultrastructural level. We conducted a comparative ultrastructural study to evaluate their morphology and cytopathic effects in cell cultures. Materials and methods: Cells infected and mock-infected with virus suspensions of Toscana, Arrabida and Alcube viruses were collected at 4 days after infection and sequentially fixed in 3% glutaraldehyde, 1% osmium tetroxide and 0.5% uranyl acetate. Thin sections of Epon-Araldite embedded samples were studied in a JEOL 1200EX electron microscope. Results: Toscana virus displays numerous extracellular virus aggregates adhering to the cell membranes and occasional particles inside the Golgi cisternae. Arrabida virus infected cells showed extensive proliferation of membranes of the endoplasmic reticulum. Alcube virus induced marked degenerative changes in infected cells. Extracellular particles from all virus infections had diameter of 76 to 80 nm and could not be distinguished by size or morphology. Discussion and conclusions: Our results show that Toscana produces much more virus particles than Arrabida but no intracelular proliferation of membranes compared to the Arrabida virus. The differences may at least in part be related to the longer adaptation of the Toscana virus to the cultured cells. In contrast Alcube virus belongs to a different species and shows strong cytopathic effects with extensive alterations of cellular organelles, suggesting that it may be more pathogenic than the other viruses.