Isolation of three novel reassortant phleboviruses, Ponticelli I, II, III, and of Toscana virus from field-collected sand flies in Italy

Abstract

Background: Different phleboviruses are important pathogens for humans; most of these viruses are transmitted by sand flies. An increasing number of new phleboviruses have been reported over the past decade, especially in Mediterranean countries, mainly via their detection in sand flies.

Results: At least five different phleboviruses co-circulated in sand flies that were collected in three sites in Emilia-Romagna (Italy) in the summer of 2013. The well-known Toscana virus (TOSV) was isolated; three new, closely related phleboviruses differing in their M segments and tentatively named Ponticelli I, Ponticelli II and Ponticelli III virus, respectively, were isolated; a fifth putative phlebovirus, related to the sand fly fever Naples phlebovirus species, was also detected. The co-circulation, in a restricted area, of three viruses characterized by different M segments, likely resulted from reassortment events. According to the phylogenetic analysis of complete genome sequences, the TOSV belongs to clade A, together with other Italian isolates, while the Ponticelli viruses fall within the Salehabad phlebovirus species.

Conclusions: Results highlight an unexpected diversity of phleboviruses that co-circulate in the same area, suggesting that interactions likely occur amongst them, that can present challenges for their correct identification. The co-circulation of different phleboviruses appears to be common, and the bionomics of sand fly populations seem to play a relevant role. Such a complex situation emphasizes the need for detailed investigations of the biology of these viruses to better characterize their pathogenic potential for mammals, including humans.

Keywords: Fermo-like virus; Phlebotomus perfiliewi; Phlebotomus perniciosus; Phlebovirus; Ponticelli I virus; Ponticelli II virus; Ponticelli III virus; Sand fly; Toscana virus.

Fig. 1

Physical map with locations of surveyed sites and position of surveyed area (red square) on a map of Italy. Urbanized areas are in gray

Fig. 2

Ultramicrograph of virions observed in the supernatant of VERO cells inoculated with ground sand flies. Round particles of 80–100 nm with envelopes and tiny projections on the surface are shown. Negative staining of NaPt 2% (pH 6.8) observed with a TEM FEI Tecnai G2 Spirit Bio-twin

Fig. 3

Phylogenetic tree of the field-detected S-segment sequences obtained by Pan-Phlebo PCR [28] and homologous sequences of Old-World sand fly-transmitted phleboviruses, obtained with the maximum likelihood method. Red: Salehabad phlebovirus species; green: sand fly fever Sicilian phlebovirus species; brown: Karimanbad virus; yellow, sand fly fever Naples phlebovirus species; sequences from this study in bold. Only bootstrap values over 70% are shown near the respective branches. Abbreviations: SFNV, sand fly fever Naples virus; SFSV, sand fly fever Sicilian virus; TOSV, Toscana virus

Fig. 4

Neighbor-joining trees obtained using genome sequences from this study and homologous sequences of sand fly-borne phleboviruses from the Old World available on GenBank: a L segment; b N gene of the S segment; c M segment