Punique virus, a novel phlebovirus, related to sandfly fever Naples virus, isolated from sandflies collected in Tunisia

Abstract

Sandflies are widely distributed around the Mediterranean Basin. Therefore, human populations in this area are potentially exposed to sandfly-transmitted diseases, including those caused by phleboviruses. Whilst there are substantial data in countries located in the northern part of the Mediterranean basin, few data are available for North Africa. In this study, a total of 1489 sandflies were collected in 2008 in Tunisia from two sites, bioclimatically distinct, located 235 km apart, and identified morphologically. Sandfly species comprised Phlebotomus perniciosus (52.2%), Phlebotomus longicuspis (30.1%), Phlebotomus papatasi (12.0%), Phlebotomus perfiliewi (4.6%), Phlebotomus langeroni (0.4%) and Sergentomyia minuta (0.5%). PCR screening, using generic primers for the genus Phlebovirus, resulted in the detection of ten positive pools. Sequence analysis revealed that two pools contained viral RNA corresponding to a novel virus closely related to sandfly fever Naples virus. Virus isolation in Vero cells was achieved from one pool. Genetic and phylogenetic characterization based on sequences in the three genomic segments showed that it was a novel virus distinct from other recognized members of the species. This novel virus was provisionally named Punique virus. Viral sequences in the polymerase gene corresponding to another phlebovirus closely related to but distinct from sandfly fever Sicilian virus were obtained from the eight remaining positive pools.

Fig. 1

Maps showing the capture sites in Tunisia.

Fig. 2

Phylogenetic tree of the polymerase gene of selected phleboviruses. Partial sequences determined in this study are shown in bold. The sequence corresponding to the viral strain isolated in cell culture and for which genetic characterization was performed (see Fig. 4) is underlined. The acronyms used in the phylogram are those recommended by the ICTV (Nichol et al., 2005): ARBV, Arbia virus; PTV, Punta Toro virus; AGUV, Aguacate virus; RVFV, Rift Valley fever virus; CFUV, Corfu virus; UUKV, Uukuniemi virus. Sequences indicated by * were kindly provided by Dr Maria-Paz Sánchez-Seco (Instituto de Salud Carlos III, Spain). Bar, 0.05 nucleotide substitutions per site.

Fig. 3

Morphological identification of Punique virus. (a) Negative-staining EM of Vero culture supernatant medium at day 6 post-infection (passage 6). Bar, 100 nm. (b) Vero cells infected with Punique virus react with anti-TOSV human serum.

Fig. 4

Phylogenetic analysis of Punique virus together with partial sequences from selected phleboviruses. Maximum-likelihood phylogeny for the S (nucleocapsid protein) (a), S (NS protein) (b), M (c) and L (d) genome segments. Sequence information corresponds to virus/country of origin/strain/GenBank accession number. Sequences representing Punique virus are indicated by arrows. Antigenic complexes are indicated on the right: PTV, Punta Toro virus; RVFV, Rift Valley fever virus; SALV, Salehabad virus; UUKV, Uukuniemi virus. Sequences indicated by * were kindly provided by Dr Maria-Paz Sánchez-Seco. Only posterior probabilities greater than or equal to 0.8 are shown. Bars indicate nucleotide substitutions per site.