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The objective of this study was to identify the phleboviruses circulating in an area endemic for sand fly\u2010borne pathogens and to estimate infection prevalence in wild sand fly populations. We also aimed to analyse co\u2010infections with\n                      <jats:italic>L. infantum<\/jats:italic>\n                      and the symbiotic bacterium\n                      <jats:italic>Wolbachia<\/jats:italic>\n                      .\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Methods<\/jats:title>\n                    <jats:p>\n                      The study was conducted in southern Spain. Sand flies were collected using CDC light traps, morphologically identified and DNA\/RNA was extracted individually or in pools. A generic RT\u2010nested PCR was used to detect phleboviruses, followed by sequencing and phylogenetic analysis.\n                      <jats:italic>Leishmania infantum<\/jats:italic>\n                      loads were quantified by qPCR, and\n                      <jats:italic>Wolbachia<\/jats:italic>\n                      was detected by PCR.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Results<\/jats:title>\n                    <jats:p>\n                      We found that 2\/443 individual sand flies (0.45%) and 8\/64 pools (12.5%) were infected with phleboviruses, with an estimated prevalence of 1.9%. We confirm the circulation of TOSV\u2010lineage B, together with Massilia\u2010like virus and Alcube virus, in three sand fly species, and report novel strains of these phleboviruses. In addition, two\n                      <jats:italic>Phlebotomus perniciosus<\/jats:italic>\n                      individuals were co\u2010infected with TOSV\u2010lineage B and\n                      <jats:italic>Wolbachia<\/jats:italic>\n                      wPrn. We also detected\n                      <jats:italic>L. infantum<\/jats:italic>\n                      in 27.6% (60\/217) of individuals, 66.7% (40\/60) of which were co\u2010infected with\n                      <jats:italic>Wolbachia<\/jats:italic>\n                      . Mean\n                      <jats:italic>L. infantum<\/jats:italic>\n                      loads [log\u2010(parasite load +1)] in specimens co\u2010infected with\n                      <jats:italic>Wolbachia<\/jats:italic>\n                      were not significantly different from those in uninfected specimens (\n                      <jats:italic>p<\/jats:italic>\n                      \u2009=\u20090.4).\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Conclusions<\/jats:title>\n                    <jats:p>\n                      This study provides further evidence for the continued circulation of TOSV and other phleboviruses in sand fly populations in southern Spain, with an estimated prevalence higher than that reported in the same region over a decade ago. Our study provides the first evidence of co\u2010infection with TOSV\u2010lineage B and\n                      <jats:italic>Wolbachia<\/jats:italic>\n                      in\n                      <jats:italic>P. perniciosus<\/jats:italic>\n                      , as well as a high proportion of specimens co\u2010infected with\n                      <jats:italic>Wolbachia<\/jats:italic>\n                      and\n                      <jats:italic>L. infantum<\/jats:italic>\n                      . Understanding these co\u2010infection dynamics is essential to elucidate the potential role of\n                      <jats:italic>Wolbachia<\/jats:italic>\n                      and the impact of\n                      <jats:italic>Leishmania<\/jats:italic>\n                      \u2013phlebovirus co\u2010infection on transmission.\n                    <\/jats:p>\n                  <\/jats:sec>","DOI":"10.1111\/zph.70086","type":"journal-article","created":{"date-parts":[[2026,8,21]],"date-time":"2026-08-21T04:38:27Z","timestamp":1787287107000},"page":"655-669","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Natural Infection by Toscana Virus and Other Phleboviruses in Wild Sand Flies From Spain: Prevalence, Genetic Characterization and Tripartite Interactions With\n                    <i>Leishmania infantum<\/i>\n                    and\n                    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