Growth of thePhlebovirus Toscana in a mosquito(Aedes pseudoscutellaris) cell line (AP-61): Establishment of a persistent infection

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Abstract
Toscana Virus, a sandfly-associatedPhlebovirus, was adapted to grow in culturedAedes pseudoscutellaris (AP-61) mosquito cell line. No evidence of virus growth was seen after primary infection of cell monolayers under maintenance conditions. On the contrary, persistent infections were established by subculturing infected cultures. Cytopathic effect was never observed. Significant titres of virus (103–105 PFU/ml), as assayed in Vero cells at 37° C, were released from persistent infected cells after several subcultures at 29° C over a period of six months. The percentage of virusproducing cells in the persistently infected cultures varied from 0.003 to to 0.017 per cent, whereas from 10 to 50 per cent of cells were shown to retain viral antigens by immunofluorescence. The virus released from persistently infected cultures did not show changes in both plaque size and temperature sensitivity from the parental virus. The virus released from persistently infected cultures multiplied in AP-61 cell monolayers reaching relatively high titres (104 PFU/ml).