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The number of newly described sandfly-borne phleboviruses has been steadily growing in recent years. Some phleboviruses are human pathogens, but their health relevance is largely uncharacterized. We aimed to investigate the circulation of these viruses in the Emilia-Romagna region where several have already been described. A total of 482 sandflies were collected in a site in Reggio Emilia in 2019 and 2020. Sandflies collected in 2020 were grouped in 21 pools with a maximum of 25 sandflies per pool, submitted to real time PCR, and isolated in Vero cell culture. Complete genome sequencing showed the isolation of a strain of a Ponticelli III virus. This virus, which belongs to the species Adana phlebovirus, differed in the M segment from the Ponticelli I and Ponticelli II viruses. Analysis performed on the genomic segments of the newly isolated virus compared with other phleboviruses highlighted a strong purifying selection in the L segments, and different substitution saturation, highest in the M segments. Future research should address the ecological processes driving the occurrence of these novel phleboviruses and their possible impact on public health. [ABSTRACT FROM AUTHOR]

West Nile virus lineage 2 (WNV-2) was mainly confined to sub-Saharan Africa until the early 2000s, when it was identified for the first time in Central Europe causing outbreaks of human and animal infection. The aim of this study was to reconstruct the origin and dispersion of WNV-2 in Central Europe and Italy on a phylodynamic and phylogeographical basis. To this aim, discrete and continuous space phylogeographical models were applied to a total of 33 newly characterised full-length viral genomes obtained from mosquitoes, birds and humans in Northern Italy in the years 2013–2015 aligned with 64 complete sequences isolated mainly in Europe. The European isolates segregated into two highly significant clades: a small one including three sequences and a large clade including the majority of isolates obtained in Central Europe since 2004. Discrete phylogeographical analysis showed that the most probable location of the root of the largest European clade was in Hungary a mean 12.78 years ago. The European clade bifurcated into two highly supported subclades: one including most of the Central/East European isolates and the other encompassing all of the isolates obtained in Greece. The continuous space phylogeographical analysis of the Italian clade showed that WNV-2 entered Italy in about 2008, probably by crossing the Adriatic sea and reaching a central area of the Po Valley. The epidemic then spread simultaneously eastward, to reach the region of the Po delta in 2013, and westward to the border area between Lombardy and Piedmont in 2014; later, the western strain changed direction southward, and reached the central area of the Po valley once again in 2015. Over a period of about seven years, the virus spread all over an area of northern Italy by following the Po river and its main tributaries. [ABSTRACT FROM AUTHOR]