Field surveys in Croatia and North Macedonia reveal two novel phleboviruses circulating in sandflies.

Sandfly-borne phleboviruses are distributed widely throughout the Mediterranean Basin, presenting a threat to public health in areas where they circulate. However, the true diversity and distribution of pathogenic and apathogenic sandfly-borne phleboviruses remains a key issue to be studied. In the Balkans, most published data rely on serology-based studies although virus isolation has occasionally been reported. Here, we report the discovery of two novel sandfly-borne phleboviruses, provisionally named Zaba virus (ZABAV) and Bregalaka virus (BREV), which were isolated in Croatia and North Macedonia, respectively. This constitutes the first isolation of phleboviruses in both countries. Genetic analysis based on complete coding sequences indicated that ZABAV and BREV are distinct from each other and belong to the genus family . Phylogenetic and amino acid modelling of viral polymerase shows that ZABAV and BREV are new members of the species and the species, respectively. Moreover, sequence-based vector identification suggests that ZABAV is mainly transmitted by and BREV is mainly transmitted by . BREV neutralizing antibodies were detected in 3.3% of human sera with rates up to 16.7% in certain districts, demonstrating that BREV frequently infects humans in North Macedonia. viral growth kinetics experiments demonstrated viral replication of both viruses in mammalian and mosquito cells. experimental studies in mice suggest that ZABAV and BREV exhibit characteristics making them possible human pathogens.